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    © All Rights Reserved
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    70 views40 pages

    Book of Vitamins

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    agarcia_290892
    Vitamins

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    of 40
    nism of TADP-Dependent Catalysis 783. Beall PHC. he Paaigm of TADP-Depenten Em -( z ‘oles of Thiamine Derive ae oat ee Role of Thiamine and ts Phosphate Desig 1.9.2. Probiotic Effects of Thiamine Derivatives in Animal Celts 7.9.3. Non-Coenzyme Role of Thiamine in Plants ° 7.9.4 Non-Coenzyme Role of Thiamine and ls Phosphate Derivatngr 7.10 Thiamine Deficiency Syndromes and Genetic Diseases Related to Thane Col Metabolism in Humans. - 7.10.1 Diseases Resulting from Thiamine Deficiency as a Primary Cause., 7.10.1.1 Dry and Wet Beribcri... 7.10.1.2 Subclinical Thiamine Deficiencies... 7.10.2 Wernicke-Korsakoff Syndrome—A Frequent Consequence of Chronic Alcoholism. 7.10.3 Genetic Disorders in Humans. Ss 7.10.3.1 Diseases Resulting from Mutations in ThDP-Dependent Enzyine 7.10.3.2. Diseases Resulting from Mutations in Thiamine Transporters or Enzymes of Thiamine Metabolis 7.10.4 Antithiamines.. 7.10.5. Experimental Models of Thiamine Deficiency .. 7.10.6 Accidental Thiamine Deficiency in Animal: 7.10.7 Beneficial Effects of Synthetic Thiamine Derivatives as Provitamine 7.10.8 Multiple Facets of Thiamine Deficiency-Related Syndromes, 7.11 Thiamine Status and Neurodegenerative Diseases 7.12. Conclusion... Acknowledgment Abbreviations... References. Begkee 7.1 HISTORY me iscovery of thiamine and its relationship tothe polyneurtic condition called berber (ake japanese isa well-known story, as it was the frst vitamin to be discovered. a particularly compe Zensive account has been given by Kenneth Carpenter in his book Beriberl, Whine ecw! Vieat B: a Disease, a Cause and a Cure (Carpenter 2000). The first description of beriberi appeared in Japan as early as the 9th century, but its relationship with diet was not suspected until the end of the 19th century. Actually, beriberi used to be a relatively rare disorder in previous centuries, until it became endemic in Japan, China, and Southeast Asia, after the introduction of polished rice t0 ‘eolace complete rice asthe staple food in these populations. In 1884, Kevetann Takaki, a Japanese dense elk beens hat supplementation of the sailors diet, based on polished sive wi com densed milk, bread, and meat protected against beriberi (Takaki 1885). As even the concept of Vitamins was unknown at that time, Takaki wrongly concluded 1 hat beriberi was a consequence of Protein starvation. At the same time, the Dutch government, in Fesponse to an alarming increase in beriberi cases in its East Indies colonies (lava and Sumatra), instigated a commission to investigate the cause of the disease. This commission, influenced by the recent discoveries of Louis Pasteur and Robert Koch, suggested that berberi was of microbial origin and cause by a toxin, ne puch physician Christiaan Eijkman, who initially was 0 memter cy ig commission, epee tne ast Indies to investigate the causes of berber. As 1 former eevee nee fe became the director of the new Laboratory for Bacteriology and Pathology in Batavia (now ‘akarta) and, in 1890, he made the observation that fowl, accidentally fed on poled eh —_ ine tha is ysis. Autopsies of these animals revealed dey resembling those seen in human beriberi (Eijk ease could be reversed after switching from generative histological chan : ges in peripheral man 1990) Inchicken, the early symptoms of ers aft g from polished to whole rice, ki tm gato Holland for medical reasons, but his fiend Adolphe Vondermancin sdk none ion fefaies in the Dutch East Indies, found that beriberi was prevalent in prisons whare the ave etry la ca ener yen fa chickens in Holland, thus disproving the hypothesis that beriberi was caused by an infectious resis that beriberi was a nutri- present in the Dutch Indies, and thus favoring Takaki’s hypoth ae Grins, hy In Batavia, Gerrit Grijns, an army physician and Eijkman’s successor, conti we Fiapicloe Mise pel nt esi eespetionerey er the absence of an essential substance that is practically absent in the endosperm but ic present inthe husk of the rice (Grijns 1901). Grijns also found that several beans had a yoni soe against polyneuritis in birds. Edward Vedder, an American army physician in Manila, was con- ‘anced that beriberi was caused by a dietary deficiency, in contrast to what many still believed. His ‘jews inspired another American chemist, Robert R. Williams, also working i ‘Together, they decided to attempt the purification of this anti-neuritic factor. ‘Atthe same time, the concept of vitamin was developed by the English biochemist Sir Frederick Hopkins, who first postulated that food, in addition to the well-known fundamental components, that i, proteins, sugars, and fats, must also contain “accessory factors” that, although present only in low amounts, are nonetheless indispensable for a normal functioning of the organism. It seems that Umetaro Suzuki and colleagues were the first to prepare a partially purified fraction from the husk of rice that could protect chicken fed on polished rice from polyneuritis, a preparation they called “oryzanin” (Suzuki et al. 1912). In 1911, Kazimierz (often anglicized as Casimir) Funk, a Polish biochemist working atthe Pasteur Institute in Paris, claimed to have obtained the anti-beriberi factor in crystalline form (Funk 1911). His claims were later dismissed, but Funk remains known for having coined the term viiamine for the anti-beriberi factor because the molecule supposedly contained an amino group. It was later called thiamine because the molecule was also found to contain a sulfur atom. The word vitamin was used then as a general term to designate a molecule required at low concentrations for human well-being. The final “e"” was dropped, because not all of these substances contain an amino group. ‘Though many groups continued to work on the identification of the anti-beriberi factor, it was ‘only in 1926 that B.C.P. Jansen and W.F. Donath, again in Batavia, obtained pure crystals of the vitamin (Jansen and Donath 1926). They sent 40 mg of these crystals to Eijkman in Holland, who could cure chicken polyneuritis with these low amounts. The presence of sulfur in crystals prepared from yeast was shown in 1931 by the German chemist Windaus working at the University ‘of Gottingen (Windaus et al. 1932). In 1933, Williams and his colleague Robert Waterman as well as other groups agreed that the correct formula was C, H,,NJOS.2HCI. It was finally synthesized by Williams (bck from Manila) and his cok leagues at Cohumbia University in New York (Williams and Clie 1936). twas fist called “aneurin” for in a 'iona -all it “thiamine” for “sulfur-cont 1g ‘rti-neuritic vitamin but it was later internationally agreed 10 cal vitamin’ its biochemical function, however, remained unknown. Thiamine can be spelled with or without the eat but. “aneurin” is now obsolete. ne Drefered, The name Taneqmeatal role of thiamine in carbohydrate metabolism were he first clues concerning the fundame hia ret fom 192 1940, Peters Provided by the classic studies carried outin Oxford by Sir RudoIPh Petes Te cof tnd associates used the thiamine-deficient pigeon 98 @ ae et vas (fed on polished rice) show tiamine deficiency. Ths isa very useful model as = Remarkably, thiamine administration atthe {yPical head retraction behavior called opis in 30 min. As no morphological brain ine Stage ofthe syndrome allows a complete e0¥6r) TT these observations led tothe ons were observed in the animal before or after thiamine FeAtnSt" the ila at that time. in the present literature, thiamine is yy QO N Handbook ay 70 en, , 1 Peters 1931). However. When the ain, lowed by the appearance of irrevering raat a li “Gaile ae as fol ntonus Was riotted within afew days. 1 esion? Already in 1931, Gavrilescu and Petey, renates from brains of deficient animals, gly Cx, decreased oxygen comsumren Ty and Peters 1931b). This decrease could be revered jt We, ccna et hat thiamine (or a thiamine derivative) wee” “ns ‘of “biochemical lesion” ne, the opist concept : st demonstration ‘ . oof thiamine, Ht was the ous rate, 1937, Lohmann and Schuser shoved te qe area em ative was an indispensable coenzyme for the oxidative decane ip TAO er cols (Lahiann and Schuster 1937), For that reason, yay ®t pyruvate, an end product of BN arongly discouraged. Later, it yeas also demonstrated tha yp” das cocarboxylase, name that is now “hs onidatve decarboxylation of another a-keto acid (2-0x0 acid), 2-orogIyany 2D coenzyme for the oxidative deca ¢ oxidation of glucose, which would explain w ThDP is indispensable for two hey bad re eiiune cromgy membre By aor! over ge ‘ been described in prokaryotes and eukaryotes, Between 195) any using the coenzyme TRDP have “rani by which the thiamine met of Than Breslow and coworkers investigated the mec Cay, « decarboxylation of pyruvate in the reaction catalyzed by yeast pyruvate decarboxylase, a Mecha ves nica of ae tao prt hme fi HC), The most important finding was that ThDP exerts its function by proton substitution OF te eee nection 2 ofthe thiazoium ring (Breslow 1958). It was also found that TRDP ig necesen eae ‘step of the pentose phosphate pathway catalyzed by transketolase. (On the other hand, ThDP was found to be produced by direct Pyrophosphorylation of thiamine by ATP, with release of AMP. The cytosolic enzyme catalyzing this reaction was called thiamine diphosphokinase or thiamine pyrophosphokinase (TPK). Thus, around 1975, the main biochemical mechanisms underlying the indispensable role THDP in intermediary metabolism were clarified. However, this general requirement of TRDP in oxidative metabolism may not be a sufficient explanation for all the known specific sympiom, appearing in the nervous system during thiamine deficiency, especially the selective vulnerailiy of yome brain regions in Wernicke-Korsakoff syndrome (Hazell and Butterworth 2005) Theefe the mechanisms by which thiamine deficiency leads to specific brain lesions continue tobe actly investigated. In this respect, we should mention the recent discovery of new diseases linked ‘mutations in thiamine transporters (Kono et al. 2009), as well as the discovery of new unsuspected natural thiamine derivatives (Bettendorff et al. 2007; Frédérich et al. 2009). It is unfortunate tht so little is known about thiamine derivatives other than ThDP and their metabolism. Therefore e shall put emphasis on these new developments in the present review. deficiency is responsible for an 7.2 CHEMICAL PROPERTIES OF THIAMINE AND ITS PHOSPHORYLATED DERIVATIVES 7.2.1 CHemistey OF THIAMINE Thiamin Chait Pyrimidine moiety (2-methyl-4-amino-pyrimidine) linked to a thizoiat Famctonal gg tonxethyb-4-methythiazolum) through a methylene bridge (Figure 7. TH evional parts can be distinguished in the thiamine molecule, * The aromatic thiazole hetey as coenzyme, ‘ocycle, directly involved in catalysis by enzymes using THP Pyrimidine he a ye active site and aids in cag ‘hich is important for recognition bythe The hydroxyeth; eee vt top linked to the thiazole ring can be esterified to form thai Phate, as well as adenylated derivatives (Figure 7.1). wwe nx A O hehe d id “O~B-O" on on on On On FAGURE71._ Structural formulas of thiamine and its phosphate derivatives. In the formula of thiamine, the ans ofthe two heterocycles are numbered according to the usual convent state TRDP, thiamine diphosphate, THTP, thiamine triphosphate; ATADP, ATATP, adenosine thiamine triphosphate. ion. ThMP. thiamine monophos- ‘adenosine thiamine diphosphates Thiamine is a white powder with a very characteristic “thiazole” odor and a bitter taste. It is auinly found under the form of a chloride hydrochloride (C,;H,,CINJOS.HCI, molecular mass 87.27 g mol) that decomposes at 198°C. Even under normal atmospheric conditions, thiamine ‘hurochloride can form a hydrate by absorbing nearly 1 mol of water. It is a hydrophilic molecule With solubility of ~1 g per milliliter of water at 25°C. It is poorly soluble in acetone, methanol, ot ‘shanol and practically insoluble in apolar solvents such as ether, hexane, chloroform, or aromatic ‘shes Pharmaceutical preparations of vitamin B, often contain the nonhygroscopic mononitrate {tem C..H,,N,0S.NO,, molecular mass 327.36 g mol), which is also used in food fortification, is prepared from thiamine hydrochloride by dissolving the hydrochloride salt in mildly alkaline ‘ektion followed by precipitation of the nitrate half-salt witha stoichiometric amount of nitric acid. 722. Stour oF THaMne 2% sid thiamine solutions are eatvely stable at room re ee oH vine 7 * ing. st ep comes heat labile. Iti therefore partially rae 1 awack on C-2 ofthe thiazolium ing PY hydroxyl attack on the C-2 of the thiazolium. See ae (Figure 7.2, II) and the MBEE*MS tothe relatively slow formation of a “pseudobase” inten Seat) war can ee oe Mts opening ofthe thiazolium ring This yields a thiolate for 0 form the so-called yellow form (IV). ctions. However, dry watch temperatures, thiamine strongly participates in See Tein is destroyed Drala ils are relatively stable, even at temperatures around JO Tt oper is “S"\letiradiation and it can easily be oxidized, particularly a L wa ' ] wo NH 30,2 on Thiochome WV) ty nie! m heterocycle of thiamine. In alkaline medion led yellow form (IV). This transformation involves the fe ening of the thiazolium ring to form a thiolate (III). Oxidation of thiamite chlyMuorescent thiocheome (V), a property used forthe experi? ermination of thi Phosphate esters. The C-2 carbon of the thiazolium ‘moiety may loses Mon yielding a highly ‘philic ylide (VII analogous to cyanide. Finally, sulfites can split Cry mednaeaC ht he methylene bee hetween the two hetensseles Yielding (6-amino-2-methylpyrimid-4) Imethanesalfonie acl (VE) an $f hyony |--methylthiazole (VIL), thiamine (1 eam be sty ‘mation of used it a highly sensitive assay, where thiamine 'S converted to the highly fluorescent thiochrome (Figure V) after oxidation in the presence of strong base, Thiamine is highly sensitive to sulfites Which cleave the vitamin between the two one tckling (6-amino-2-methylpyrimids-yDmeth, tesulfonic acid (V1) and 5-B-hydroxyethy!+ methylthiazole( VI) (Leichter, ‘and Joslyn 1969), As sulfites are: ‘widely used as food preservatives, this Feaction can be responsible for thi nine cleavage in food during storage, even at low temperatures. Finally, the thiazole ring can bse its Cs hydrogen, forming an ylide (Figure 7.2, (Vil). This von is highly unfavorable in aqueous s olutions, but the ylide is an essential intermediate if ThDP-catalyzed reactions, where its formation is favored by the conformation of the catalytic sit€ of the apoenyme, 273 he C-2 proton is very high (in the 14 we 14-20 range ea otc ange), the ylide carbanion is neve ca hi.) pt Nt sng wok mig 2 pron ix readily demonstrated by its rapid ex hang: px, oft fous soluti ip relative ami PROPERTIES OFTHE Tuurzoue Herrrocvait 33 123 Ronald Bresiow in the 1950s, the catalytic properties of thiamine result from th lamine result from the fhe planar thiazole heterocycle ty of i rycle (Breslow 1958), Thiazole is rare! od to oxazoles and imidazoles, the sulfur atom, replacing either an oxy roles are relates 7 s sible for a larger deloc trogen atom. is fespons/le [A il Jocalization of n-electrons th 4 z ‘electrons than in oxazoles oF ne acidity ofthe C-2 proton, facifitating the formation ofthe gids car is ine Sifu, The alkylation of the N-3 ofthe thiazole heterocycle 0 form a thiazolium cer of C-2, thus stabilizing the carbanion asa yl. AS ae ae rs an analog of cyanide, able (0 catalyze benzoin condensation. However, fre thine oxo acids requires an enzymatic envi- carbaniot F Mieis poor cata its action in the decarboxylation of sent ha cea ates the reactions by a factor 210! (Kluger and Titman 2008) 724 Cemicat PropeRries OF THE PyRiMiDiNe HETEROCYCLE pyrimidine heteroeyele is Jess reactive than te thiazole heterocycle. The 4-amino group WY psicharactr and itis NOt protonated even in strong acids. However, js important for positioning Mecoenzyme TRDP in the active feof the apoenszymes and it is required to form fluorescent thio~ a oe snown below. The N-I'is protonated at pH 3 mg/kg). Cereals, mainly whole grain cereals, also contain relatively high amouns the vitamin, Moreover, many breakfast cereals are fortified in thiamine, On the other hand e- tables and fi poor sources of the vitamin as vegetables contain two to five tins jess thiamine than meat per unit weight (Davis and Icke 1983). While thiamine js stable on song. it should be emphasized that food processing may have an impact on the final thiamin cone. As vrentioned above, thiamine is heat bile, and therefore, overeooking, pasteurization of mk heating of canned food may result in considerable loss of the vitamin. Nowadays, many processed foodstulf such as cereals, bread, dairy products, and infan fore tas are enriched in thiamine as well as other vitamins such as niacin, riboflavin, oF folic acid, tis generally considered that the recommended dietary allowance (RDA) is 12 mg/day for adult mas tind LL mplday for adult females (Table 7.), The RDA is inereased to 14 mg/day during pees its are relativel TABLE 7.1 RDAs (mg/day) for Thiamine in Humans Males Females 12 MW ld Lactating Ls Child, 1-3 years os 0s Child, 4-8 years 06 06 Child, 9-13 years 09 09 Adolescent, 14-18 years 2 Lo. Note: RDAs were according to Bates (2007). - ee pon __ 15 mptday for lactating women, tn hi ae ean LS ildren, the bene andy RA a hg i itnsome cation as they depend on liesye ayy ae sho homers aise Ould however he vores thiamine supplementation is advisable z, as both might have destnsed ine ie a stina aerequite large thiamine intake (100 mg/day orally or i fopathy and Some FARE Benetic diseases such a6 ene er) or thiamine-responsive maple syrup urine die lly in humans), derivative o amine, or fursul anal sultiamine; see Mperapeutc ses May Vary From 1010200 mila. Rory severtl days are generally recommended for the treatment of cardi jadi that intravenous thiamine administration precedes intravense ata Met beter. I pelater may worsen thiamine deficiency (Hack and Hoffman 1998) Ingo nn taton as tht igh carbon uptake increases the requirement for thiamine, posibh Sear pot enzyme-bound TRDP during the catalytic reaction (McCown intake would result in increased flux through ‘ThDP-dependent enz} mes {pwn of THDP and worsening the thiamine status, tts ‘Thiamine has a very low toxicity and is without adverse effects after oral intake of ppallergic reactions (anaphylactic shock), respiratory depression, and neuromuscular blockae, In dos, blood thiamine levels of 10 mg/100 ml (0.3 mM) ate invariably fatal (Davis and leke 1983) prtly through a curare-like action of thiamine (Ngai etal. 1961; Smith eta. 194) a abusers and might be nie ‘might be useful for absorption, Some Sly). These include Wernicke's Ponsive megaloblastic ane ‘use OF the slow rates of thi lability than thi on 7.10.7). 100 mg thiamine intravenous I ase, Be S with higher bioavai mine ne may be of For instanc sn observed ‘result of an instabile al, 2006): increased glucose s, hence precipitating break- 74 ANALYTICAL PROCEDURES AND DETERMINATION OF THIAMINE IN BIOLOGICAL SAMPLES AND FOODSTUFF Early methods for thiamine determination include microbiological assays, thin-layer chron phy, electrophoresis, and low-pressure liquid chromatography. Since the 1980s, high-perfor liquid chromatography (HPLC) has become the method of choice for the separation and quantifi- ‘ation of thiamine derivatives in tissue samples, food, or pharmaceutical preparations (Kawasaki 1992). In most cases, detection involves transformation of thiamine derivatives to the corresponding fluorescent thiochrome derivative (Figure 7.2). Quantitative derivatization is obtained after oxida tion with potassium ferricyanide or cyanogen bromide in strongly alkaline medium, Thiochrome detvatives are highly fluorescent at pHi >8 with an excitation maximum argund 370 am and an mission maximum at 433 nm. oan Most HPLC methods rely on reversed-phase columns with or without fon-palemg kt il tre based on either precolumn or a posicolumn derivatization procedure (Baten at 1851; Fayol 1997; Kawasaki 1992). We prefer precolumn procedures for several reasons. First, : thiazole ring, ic) ing the positive charge on the U fave a more hydrophobic character resulting i - sence of Responding thiamine compounds. Second, postcolumn derivatizatic eeu ow action coil increasing void volume and resulting 1 decreased res H, conditions under which ta derivatization is that thiochromes are only fluorescent at alkalis PT tine pl uy sca hased columns are unstable. Alternatively, Sone) 1°27 Ngo, poatemps ea 3 polystyrene-divinylbenzene resins, can be used — ould be interesting alternatives '2). Nowadays, relatively alkali-resistant silica-base fon ide-based phases has beer described Plysiyrene-divinylbenzene resins. Recently the USE OLN covery of he new a I. urea 2005, Pinto etal. 2002) However coms are, inane ro name thiamin derivatives, the only method documented Un NAD 6 rad ATREP is the Jon-palring method that we py, ty et al, 2009) ive and amounts as LOW as seve Trenty scotive ak! amounts 38 TOW 88 NEFA emgga fe or its derivatives at nanomolar conces eMttatiog, SP ion, he she i pornpe, TEP, ATRDP. yt ‘ oot, Frede (Bettendortt etal b Mast of these methods et atlewang the detection oF Mami det SYNTHESIS, DEGRADATION, AND 7.5 THIAMINE BIOS . Y RIBOSWITCHES REGULATION B' sharyotes, fungi, and plants by complex pathways (Jur, ive synthesized separately and then, on ay mine (plants, yeast) or phosphorylated to ThDP a hes ty thiamine monorphosphate Kinase (EC 274.16), The thiazole moiety is synthesize ja Mia heeraldchyde 3-phosphate. The synthesis of the Pyrimidine? ine phosphate, derived from the purine biosynthetic pathy survival of most known organisms, the enzymes ina, biosynthesis are potential targets for antimicrobial agents (Du et al, 201), ‘howynthesis occurs in chloroplasts by pathways similar to those de ii the ThMP formed is hydrolyzed by relatively unspecific mona! {Aji etal 2007b; Julliard and Douce 1991: Ra la-Kozik et al. 2009). Thiamine istheneiness aaa towol, where i is pyrophosphorylated to THDP by plant TPK (Ajai etal Oat Ho papete nt corplasts, where itis the coenzyme of transketolas inte yyy jn etal. 2012). hin Many organisms cont ading enzymes (thiaminases). Thiaminase I(EC25)3 vnicnorganisms and some higher multicellular eukaryotes such as fern, shel a ise, Hence, consumption of raw parts of these organisms by animal ¢ ial thiamine deficiency syndromes, such as beriberi in humans or cor physiological significance of this enzyme is not known, The il-anst aminase IT (EC 3.5.99.2) is a hydrokase, not involved in the breakdown of thiamine as previns) the hydrolysis of aminopyrimidine to hydroxypyrimidine, a building st ia (lenkins et al, 2007). Thiaminase Ii thrive sized in Thiamine ts sy 200), The thiazole and py THAIP, which is either hydrolyzed to th +. hyalroxymethyt pyri’ ne is probably required for the Obed nts, hia in bacteria and yeast nt in t a pyrimidine transf man leads to sometimes necrosis in ruminants. The for the biosynthesis of thiamine by some bact considered part of a thiamine salvage pathway The degradation and elimination of thiamine have not been extensively studied. After admis h doses of thi ine, the excess is eliminated, mainly intact, by the kidneys withins few hours. It seems that thiamine can also be degraded in mammalian cells, but no enzymes se cifically involved in thiamine degradation in mammals have been identified. [tis therefore probate that most of these degradation products result from the action of several detoxification zm present in the organism, When radioactive thiamine is administered in rats, many degradation pt tucts appear in the urine, notably 2-methyl-4-amino-5-pyrimidine carboxylic acid and 4-net thiavole-S-acetic acd, resulting from the cleavage between the thiazole and the pyrimidine OF ( and Pearson 1964a,b). Riboswitches, a relatively new concept (Baird etal cifically binding small ligands in order to regulate gene expression. Typically, one of the end ucts of a biosynthetic pathway binds to the mRNA coding for a protein early in the pathway te regulating its expression. The binding of the metabolite triggers an allosteric change in dimensional structure of the mRNA, affecting protein expres lation, or splicing, Until now, riboswitches have only been found in bacteria, Fung 2 ig notin animals. The most commen ligands for ribaswitches are THDP ‘S-adenosylmehion 2 “n pelea T pa hbo) is widespread in bacteria, fungi, and pa ending of THDP tthe tbo induses the formation ofa terminator hairpin, had ‘everal thiamine biosynthetic enzymes. The mechai different in fune! 1, 2010; Winkter et al. 2002) are mRNAs 4 a7 involved in the control of RNA s ki Is sat lve control of RNA splicing rather than transcription or translati ion or translation. box is i nly riboswitch so far described in eukaryote: thi he Hr pgwitch is the OF tT seep ribos METABOLISM AND DISTRIBUTION OF THIAMINE DERIVATIVES 16 “if MecHianiss OF THDP SyvtHesis 16" : “above. thiamine Biosynthesis in prokaryotes fi erie 2 imine rryotes, fungi, and plants yields rather ase hia. 1 hacteria, ThMP is phosphorylated by ATP a ee ned wep kinase (0 eld the coenzyme TRDP. In plants and yeast, THMP is frst hy Mrolyed 0 ce a ins © es esponsbe ortisydrasare not kon we be dee belo ees ie mmonophosphatase CToMPas) has been characterized so far in either ‘rok cies Syaryotes: HOWEVEE several nonspecific phosphatases are able to hydrolyze “Thiamine oreo en DE yrophosphorylated by ATP (thiamine + ATP 3 ThDP + mar Ar ration oe thiamine diphosphokinase (TPK, EC 2.7.6.2), also called thiamine pyrophosphokinase i 4 yet eg soluble, cytosolic and ubiguitous enzyme, except in enterobacteria such as E. coli and i in other bacteria such as Paracoccus denitrificans (Sanemori and nla species: Put Pe Kavasaki 1980) and villus subsiis (Melnick etal, 2004), In the later, which lacks thiamine for thiamine salvage. 139 (Banga etal, 1939) and first character- wasaki Kase, TPK is important Jhasovered by Banga, Ochoa, and Petes in 1 ally purified from baker's yeast (Kaziro ¢ terson et al. 1975). Complete puri (Gubler etal. ritrificans (Sanemori and Kawasaki {Voskaboyer and Ostrovsky 1982), and human red blood cells (Exi et al dependent enzyme. The apparent Kx for thiamine is very low (0.I-1 #M), but for (estosolic) Me" Fp itis of the order of 10 mM. In Yeast. GHP seems to bea beter substrate than ATP (VoskooyeY (8,0 109.5). The TPK gene has been iso- ‘adOstrovsky 1982). The pH optimum broad and alkaline ined and characterized in Saccharomyces ro pevisiae (Nosaka etal 1993) and Schizosaccharcnye (Fankhauser etal. 1995). The sequences fof mouse (Nosaka et al. 1999) and human (Nosaka fort 700K; Zhao et al. 2001a) TPK became know shortly afte, followed by the crystal structures of arouse Timm etal 2001) and yeast (Baker of 31 2001) enzymes. According 1 these data, TPK isa homodimer of 46-56 kDa binding to Motecules of thiamine at the interface of ‘each subunit. pane a recent study described a new etystal tryeture of mouse TPK forming a tetrane (Liv and Hurley 2011). The human enzyme has properties similar to those described for the rat and pig brain (Bettendorff et a the catalytic centers between ‘mammalian, bacterial, |. 1996). Differences in " a and fungal forms of this essential the possibility of designing drugs with differential I enzyme rai fects on these enzymes that could be used as anvil ‘TP. rather ubiquitously expressed in most OE of function mutant showing decreased physiological rhy' pans de Jong etal. 2004) tis present inal Drain Ata. 1986), the highest activity being found | mexebral cortex and mira. In burns ~~ sive ‘exons; the highest expression is veralthiamine antimetabolites ae Poten. Prianine = 1 uM wih eee © porn) is a substrate Of TPA tno {seal 2006) On the othe ha ‘thiamine Hs fect an inhibitor as reiting (Rind: etal. 1963). Oxsthiane a a se (Datta and Racker 1961) as well as of eS th tt . " = ThDP + Al hasbeen shown thatthe equilibrium of the TERT wna oe “ah pecause of bin left ft (Peterson et al, 1975). However, in viv: u al, 1961), rat brain Johnson and Gubler fication was later achieved from pig brain 1980), rat liver and brewer's yeast 1992), TPK is a soluble was ined and part 1968), and pig brain (Pet 1982), P. de robial agents. ‘ams, except enterobacteria. A partial loss- ithms was described in Caenorhabditis ele- vestigated (Matsuda etal. 1985; Rindi the lowest activities Were the TPK gene mosome 7434-436 and i jet al. 2005). ipstrates of TPK. Thus, not very efficient but a better sub- inhibitor of tran- ompettive inhibitors oF SP MP is farto ding 278 or transport into mitochon Jex (OGDHC). This probably ex of newly formed ThDP to transketola wrate dehydrogenase comp! cytosolic ThDP accounts for only approxin al (Bettendorf etal, 1994b). However, the proportion of free cyte fther organs such as fiver tas also reported that admin stration of high di Cursors with high bioavailability leads to increased THDP levels in liver and red fy can therefore act as reservoirs for the vitamin (Volvert al. 2008). How such an acu TDP is compatible with a K,, € 1 is not understood. In organisms such ay cal why synthesized by phosphorylation of ThMP, the fre (Makarchikov et al, 2003), and oxogh 7.6.2 MECHANISMS OF THTP SyNTHESIS 76.2.1 Possible Involvement of a ThDP:ATP Phosphotransferase— Hypothetical Implication in Leigh’s Transfer of a phosphoryl group from ATP to 1 tion ATP +-THDP + np, ThTP alyzed by a “ThDP kinase” (EC 2.7.4.15) would be the most obvious mechanism fay THTP synthesis. The presence of a soluble, cytosolic ThDP kinase was indeed reported in exta, from spinal cord (Eckert ‘and Mébus 1964) and rat liver (Voskoboev and Luchko 1980), In, 194, h et al reported the complete purification and characterization of w THDP kinase fn brewer's yeast (Chernikevich et al. 1984). However, the enzyme had a very low speciticatvty ant it is not clear whether the reaction product was authentic THTP (we later found that ATHTP migh also be formed and misidentified as THTP under the conditions used by Chernikevieh et al; se Makarchikov et al. 2007). In the mammalian brain, the mechanism of THTP sytihesis was inv tigated by Cooper and associates at Yale University. They first suggested the existence of a ThDP kinase in a crude mitochondrial fraction (Htokawa and Cooper 1968). However, ates to cha terize the enzyme in a soluble form were unsuccessful. Finally, Nishino et al. reported the pores tion of a kinase from rat brain, but the substrate was not free ThDP (which was not phosphorylated, but ThDP bound to a protein that they isolated from a rat liver cytosolic fraction (Nishino et al 1983). The nature of this ThDP-binding protein was never clarified. According to Voskoboey and Chernikevich (1985), the only ThDP-binding protein in liver cytosol is transketolase, and theres no evidence that the latter may be involved in ThTP synthesis (Voskoboev and Cher rnikevich (9X5) In any event, the kinase isolated by Cooper and associates had a very low specific activity an tho results were never reproduced elsewhere, Actually, using recent and reliable HPLC methods © imate THT, we, and others, were never able to demonstrate the occurrence of any sole THF ity in brain extracts. Likewise, no such activity was found in electric organs, pig bal or cell-free extracts from E. coli although high amounts of THTP can be accumulated in those and thiamine triphosphatase (THTPase) activities are negligible (Bettendorff, L.. and Makan ‘A. unpublished result Yet, early data concerning THIP synthesis in brain should be briefly analyzed here, claimed to be relevant tothe diagnosis (and possibly the etiology) of a rare neureegene'as ie ease of early childhood aed subacute nerotizing encephalomyelopaty (SNE. Lah shart Denis eighin 1951 Fister 2008 Leigh 19S), Although the foi tissue mec Wer Oe ditferent areas, Leigh noted some similarities wih sions found in Wernicke Korat ‘0 thiamine deficiency. Disturbances in the oxidative decarboxylation of PY. ‘ThDP-dependent process) were indeed found in aut NNN etal, 1979), Actually, high-dose thiamine ati this was observed for ess than 10% of ca An alternative hypothesis was ease was linked to activity. They DP through the 1 Chernikevi avit wes cl topsy samples from patients with St treatment was found to be beneficial for some (Di Roceo et a. 2000; Naito etal. 1997) eds pothesis was proposed by Cooper and associates, who suBgsst gy reported that te er LEP production in the brain, rather than to reduce te Ported that the THTP content of the brain was markedly tower if P sain nist pions With SNE, whi jn the Fy “oaeamine Comer ta. 1965: 2 aacte ane spina ak 8nd Ueprotcinized Hog pees: 197) In aon it wos ey m Was claimes ras according 10 the reaction re ati “paaesis accor Feaction reported ab atients contained an inhibitor of TTP then, no further data appeare Ve, using protein. I Coarcand 8 cnt he Tea UNI THDP exacted fm ne jaa porte and colleagues might res 'S and it has been sug, worth ight re ul from a chromatographic ee some Inthe author’ laboratory, no THEE petendortt et G 16) in contrast to ae seause it is hydrolyzed by the soluble 25 kDa SSxDa THTPase remains ative in brain ao pase jnastoma cell (hich have alow THTPase ativayy ie Postmortem. In cultured mouse new. {visy this would be compatible withthe disappearance ne trust TATP was ~I h (Bettendorf Cooper and coworkers (as well a other investigators in eatin ees Postmortem delay THTP concentrations (even postmortem) were as high as 10% oy Ne fePorted that brain fret tis have consistently shown that the amevs are tft thiamine, ile our mare tea hiamine (Bettendorf etal, 1996; Gangolf etal. 2010a) is thes a owe than 1% of idenitied as THTP by Cooper and colleagues was not authentic THEP wee ona the compound aswell asthe reason why it shouldbe absent inthe brains of pation wig ne com anintriguing enigma, Concerning Leigh's syndrome, it istow considered that igh’s disease, remains deficits in mitochondrial respiratory chain complexes I, I Illand 1V se elle hore ‘HOP-dependent PDHC would be affected in a small percentage of paiene he Gnuck supplementation could be beneficial (Barnerias etal. 2010; Di Roceo eta mete on, be detected in ostmor ed hain sa Postmortem human brain with SNE * (Gangolf etal, 2010, al, 20104), presumably uring the postmortem delay. Indee 16.2.2 THTP Synthesis by Adenylate Kinases “Athough THTP generally accounts for less than 1% of total thiamine in animals, ina few tissues such as pig skeletal muscle (Egi et al. 1986), Electrophorus elecrricus electric organ (Bettendorf etal. 1987), and chicken white muscle (Miyoshi et al. 1990), THTP is the major thiamine compound, accounting for 370% of total thiamine, Kawasaki and coworkers have suggested that this high THTP content was cor- related with the high amount of adenylate kinase (AK1, myokinase, EC 2.74.3) found in skeletal muscle and probably in electric organ. Indeed, these authors (Shikata etal. 1989b) have shown that, in vitro, AK may synthesize THTP according to the reaction ThDP + ADP + THTP + AMP. ‘Though this reaction is much slower than the normal reaction catalyzed by AK, that is, 2 ADP [ATP + AMP, Kawasaki and coworkers provided convincing evidence thatthe former reaction was responsible for TTP accumulation, both in pig Shikataet al. 1989b) and in chicken Miyoshi etal. 190; Shioda etal, 1991) skeletal muscle. This is surprising, as AKI has a sharp pH profile with an optimum at 10 when THDP is the substrate (Shikata etal, 1989a). Moreover, the K,, for THDP is high (0.83 mM), vell above the usual cytosolic concentration of ThDP, which appears to be generally ower than 10 HM (Bettendorffet al. 1994b). On the other hand, we have shown that THTP levels in adenylate kinase knock- aut mice are the same avin wildtype animals, Furthermore, E.coli els are able to rpidly accumulate high amounts of THTP after heat inactivation of their only AK isoform. These experimen demonstrate tha other mechanisms for THTP synthesis must exist in both prokaryotes re at In conclusion, itis likely that in skeletal muscle and most probaly ine ee 0 reaction responsible for THTP synthesis isthe one catalyzed by AK). TW A Cytosolic enzyme, is in agreement with the ‘observation that THTP is Sere aan Of skeletal muscle (Epi et al. 1986; Matsuda etal 1989) and eles sn be imerpreted Eder and Dunant 1980). The accumulation of larBe amounts of en ll beshied to terieht ‘flows: the equilibrium ofthe eaction ADP-+ TADP-S THY Ti eaton are catalyzed because AMP reacts with ATP (which is in excess) eee ADP. This equilibrium will also BA te pt eatin wil Be TOE gree a ie shifted to the right because the muscle contains Mig! 780 Handbog, Vy, kinase: ADP + phosphocreatine = ATP + creatine. The global process ¢, oe aN thus be hn, reaction ThDP + phosphocreatine +> THTP + creatine. eh, 7.6.2.3 THIP Synthesis by a Chemiosmotic Mechanism cs In the 1990s, we reported a net synthesis of THTP in «particulate Fraction ¢ isolated from rat brain (Bettendorff et al. 1993a,c, 1994a) with either thiamine ort} Meu, THTP was formed inside closed compartments that we thought to be itt is real reinvestigated this hypothesis and we showed tht THTPis sythesized in rg Ree chemiosmotie mechanism (Gangolf etal. 2010b). THTP synthesis requires intacr oto functional respiratory chain. Iti inhibited by uncouplers and by inhibitors off mien ing that it might be synthesized by a mechanism similar to ATP synthesi oP rATPa a sing _ Phosphorylation and driven by the protonmotive force: ThDp + P, ao Processor ATHTP synthesis was observed in isolated mitochondria from en of substrates ofthe respiratory cain. Addition of external THDP ted to am inn thesis probably because THDP eters mitochondria via specific tansponer ees MP ‘membrane of the organelle (Lindhurst etal. 2006; Marobbio et al. 2002), nthe ine External phosphate activated ThTP synthesis and it also incre: mitochondria. tis not known whether his release i of physiological imporce fest that THTP produced inside mitochondria plays a role either in the interme the eytoxol (Figure 7.3). When brain slices were incubated with the uncouple decreased, suggesting tha the chemiosmotic mechanism is operative in vee Benous ThDp; ased the release of Tyrp from uti iy -mbrane pa at Cece Tipe Thiamine io a mammalian cell. oe 7 s synthase); 5, adenylate kins iné Monophosphatase; 4, THTP synthase (could. a“ ATP and ADP as sabatrate 8 7 Ee thiamine triphosphatase; 7, ThDP adenylytransfersse oa hydrolase: 9, thiamine transporter; 10, mitochondrial TDP erie wor it - 4 mechanism would account forthe fact that in the mammalian brain, such a tochondria. Interestingly, no such side sy! le levels of THTP. The arly undetectabl ae THTP synthesis and a tight regu Ee nent with results obtained in E. coli, whe '* synthesized only in the absence of ia ePin te incubation medium, provided that a suitable carbon source i, resent (Lakaye si yin the absence of a carbon source, they symhes ATHTP y al sal. 2010), (Betiendorff. et al. 2007; aa a 463. MECHANISM OF ATHTP Syyriesis 16: has been shown fo be sHthesized in vttoinE: col supemtans by a TRDP adenylyl. ee according to the reaction TRDP + ATP (ADP) == ATHYP PP|(P.) (Makarchikov etal Sp07) Both ATP and ADP were substrates for the EAZYM. Which aso required a divalent cation sh Mp of Mr as well 28a heat-stable soluble acthonns enzyme is probably a high ‘olecular-weight multi-subunit complex. | 164 Baenanc HyoKOWss OF PHostrontareo Desivarves oF Tuqung Thiamine phosphate derivatives are hydrolyzed by many nonspecific phosphatases, in particu- lar alkaline phosphatase and acid phosphatase. The latter 'S sometimes used to hydrolyze thia- rine phosphate esters in biological samples to generate thiamine Which can then be measured amd expressed as total thiamine. It is known that in vivo THDP. the biologically most active thia- sine compound, is hydrolyzed to ThMP, which in turn is hydrolyzed to thiamine, but no specific guys hydrolyzing those compounds have ever been described. In contrast, a highly specific TATPase exists in animal tissues, 7641 Thiamine Triphosphatases (EC 3.6.1.28) A number of phosphohydrolases such as my 'yosin ATPase (Murai and Katsura 1975), side triphosphatases (Nishimune et al. 1987), and mammalian acid or alkaline (Penttinen and Uotila 198] iydrolysis of THTP with variable degrees of addition, two THTP-hydi have been found in mammalian tissues: a fe mate -ssociated enzyme. Only the soluble enzyme has hee characterized and p ‘HTP. E. coli nucle- roven specific ‘The brain mer re effective i $™M for Thop. ‘The nonhydrolyzable methylene phosphonate analog of THTP is a weak competi- ‘We inhibitor {K;= 3 mM). Though the inhibition of THTPase activity by ATP was only partially mPetitve (Barch; 1976), those results suggest that the active site may have an affinity for ATP vide ebstantially higher than that for THTP. The membrane-associated THTPase ‘might thus be an ntified nucleoside \iphosphatase rather than a true THTPase. So far, it could not be identified | acoaa ktown membrane ATPase. It is interesting to note that histochemical evidence suggests ‘alization of Soluble and membrane-associated THTPase in brain, where they are associated spe pres¥haptic membranes, synaptic vesicles, and Golgi apparatus Ogawa etal, 198 PE eS Of the enzyme in fish electric organs and mammalian skeletal muscle difer That f° other tissues (Bettendorff et al. 1988, 1989a; Matsuda et al. 1991). The dis- “ature of THTPase in muscle and muscle-derived tissues is its strong activation by > > en “a: Bi te Ie re 282 ONVitanin, SCI), an activation not observed for sibly inhibited by the anion the from E. elec reversi oo M transport inh rit erage The bene 2.2-dslie i (DIDS). Anions protect agains inhibition pei spartiolat, SO! ions were effective ‘at 5 mM. This suggests that an anion-binding site i ig, in particular, SOs Mrition of the activity. The substrate also protects from inhibition by yn for catalysis oF Tee! protective effect was 0.25 mM. 8 ang ‘on constant estimated from the oe eectricus en7y™ could be solubilized by various neutral detergents but the acti ost during purification (Bettendorf et al. 199la); is also been observed previously vie i \d Braun ). 7 brane-bound brain enzy! anc Z goa ‘pamerous studies, the role of membrane: sociated THTPase remains unclear. Asi, nora obtaed in pure form, is specificity for THTP Nerney unproven and its catalytic eg se pabably low: the estimated K, vues range from 0.25 mM in E. electric organ Betensn in rat brain (Barchi and Braun 1972b). Those values ate well above i al, 1989a, 1991a) to 1-2 mM it : physiological concentrations of THT in most AT Itis thus doubtful that membrane-associany ‘ThTPases could play a signific the regulation of cellular THTP levels. In contrast, the soluble THTPase discovered ‘by Hashitani and Cooper (1972) was found to more interesting properties It was later purified to homogeneity (Makarchikov and Chernikevich 19 ‘and sequenced (Lakaye et al. 2002), and its three-dimensional NMR solution structure has been dy ined (Song et al. 2008). The protein was found to be a 25 kDa monomer. The activity required Mg ions while Ca?* ions were inhibitory (ICs = (0.2 mM) and the pH optimum was alkaline (85-95) Thy enayme was highly specific for THT: neither nucleoside triphosphates nor THDP were hydrolyzed. The K,, varied between 15 and 150 }M depending on the species, and the catalytic efficiency was very igh GK, = 09 x 108 0 25 x 108 x" MD, The THTPase MRNA is wi idely expressed in human tisus, though at relatively low levels (Lakaye et al. 2002). Immunocytochemical studies and in situ hybridize tion revealed that, in rodent brain, the enzyme is mainly expressed in neurons, with a prominently cy solic localization and a possible nuclear localization in glial cells (Czerniecki et al. 2004). Th 2002, Iyer and Aravind pointed out that the catalytic domains of human 25 kDa THTPse and CyaB-like adenylyl cyclase from Aeromonas hydrophila (AC2, [Sismeiro eal. 1998) dene novel superfamily of domains that, according to these authors, should bind “organic phosphates” ‘This superfamily was therefore called “CYTTH” (cyaB-thiamine triphosphatase), and the peseks = A Tanmehs of orthologs was demonstrated in all three superkingdoms of life. This suggested that CYTH sat salt ( ancient enzymatic domain and that a representative must have been present in the last universal = common ancestor of all extant life forms. TL must be emphasized that 25 kDa soluble THTPase activities were observed only in mans with the exception of the domestic pig (Makarchikov et al. 2003; Szyniarowski etal. 2005) Inet, in the latter species, several point mutations make the enzyme highly inefficient, No significant sole ‘ThTPPase activity is observed in bird tissues and there is no evidence for the existence of a gene coins for the 25 kDa THTPase in birds. This explains why THTP levels can be very high in fshelectic oP (Bettendorff et al. 1987), bird (Shioda et al. 1991), or pig skeletal muscle (Egi etal. 1986). Further: studies involving human tissues and cultured cells suggest that the 25 kDa ThTPase plays a major the control of cytosolic THTP levels that are generally low in mammalian tissues (Gangolt ¢ al 2008 N- > NO; > Br ¢ anions (I >SCI chaotropic anions (I 7 me (Barchi a cesta amin 7.6.4.2 Thiamine Diphosphatases (EC 3.6.1. -) Enzymatic hydrolysis appears to be the main metabolic fate of ThDP in many tis a some cell types a significant amount may be converted to THIP or ATHTP. ‘Therefore. ee terization of ThDP-hydrolyzing enzymes is an important issue as they determine the eile essential coenzyme. vt In the early 1960s, thiamine diphosphatase (or thiamine pyrophosphatase, ‘ThDPase) aig reports to occur in microsomal nucleoside diphosphatase (NDPase) preparations oe lian lver, Yamazaki and Hayashi obtained a nearly homogenous NDPase preparation aga) AT 3 microsomes, also exhibiting a significant ThDPase activity (Yamazaki and Hays sues, toh? mr 283 both activities by an allosteric ‘hanism, we and dUDP, followed by THDP and Gp, tree PIERS activity wag obtained with IDP, GDP nal enzyme did not hy ores toa significant extent. While the Sloman ‘von fea hydrolyze Ap Por nucleoside ound 7, it was around = In the presence of ATP as 3.5 mM for THDP and 0.63, enzyme in TADP hydrolysis, ais brain (Barchi and Braun 19724; Iwata et al. . al and microsomal fractions and it wae sug: that this enzyme is identical to NDPase, but in contrac 0 liver NDPase, it was iahoned sa acivted by ATP. The pH optimum was alkaline and it requ a dival Independently of these biochemical studies, ThDPase activity has ker of the Golgi apparatus (Goldfischer etal. 1971) They evil at neutral pH. Histochemical studies , é ¢ retic liver cells, with a neut endoplasmic reticulum in TaDPase activity. ATP stimulated the activity of the endoplasmic ann ent cation as activator. been used for a long time Vity of the Golgi ThDPase . Sano et al. reported the existence in rat brain of two types of NDPases hydrolyzing Some wah Properties similar to the microsomal liver enzyme, was called L-type and the other eta ear 1984). While ATP activated the former, it inhibited the later. The rs . Ret Probably corresponds to the ThDPase of the Golgi (Sano et al. 1988b). Type B ene purified fom rat brain after ‘detergent solubilization (Sano et al. 1988a), and the purified Eee anand TADeCOr DP and UDP without preference. The Kas L06 mM for TADP eee ati divalent cation with the preference Mn* > and slightly below | mM for NDPS. The activity required a dival eee sacJmctclr mass nar 1S EDs. However socal tanh were chenc seinen Rey ie ee a ree eee jing the existence of isoforms (Sano et al. inplyacrylamide gels afer ing! activity saining,sgpesing the xine Rearmed 18, IB) The B-type NDPase most probly corresponds Deboing wks ATPase supetuny putative transmembrane protein of 610 amino acids ( Soe ed eps vee cations ieee ad ina se ae cea ee aoe neath ‘on the luminal side and can only rs Mg?" > Mn). It is localized in the Golgi with the act ‘other UDPase, also hydrolyzing GDP Semel ith detergents (Trombetta and Helenius 1999). A ee aly rang a B eoeated ¥ inthe endoplasmic reticulum and elon 0 prin fami drying eb peel ponds in nucleoside di and phosphate oor eialum of mot maralian it men of the endoplasm n, Indeed, while eee ‘hi pa with Lye Teas erDPae negligible ty only The question of the identity of t Se eadpinnie regu UDP et ‘authors state that the ThDPase activ Mopar ie ty h L-type (ested the activity at pH 7.5, a pH Geren ‘and Hayashi eee sn, the ThDPas activity o effective in hydrolyzing ThDP than iched fractions o ga Dee mnal and glial cell-enri afore etal permintn ceeuionct seas higher in the neuronal ration (ty jt i PH 9 was found to be 15-fold hig! sted that the alkaline L-type tells (Mastrogiacomo eta ees Of ThDPase in cultured cells sugge: is found in neuroblastoma aL 1979: Martinez-Rodrigu net Tas ile the B-type enzyme i (ez Murillo ot at and glial localization fr This confirms earlier histochemical st ting a preferentially new a ‘1 1978, 1980; Ogawaeet al. 1976). ue Jum and Golgi Ss An Band Ltype enzymes, Pc poth endoplasmic reticl pecs of aboot cue thatthe role of bah

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