You are on page 1of 5

Glycolytic Pathway in Rumen Microorganisms

Dwayne Hamar and Raymond Borchers


J ANIM SCI 1967, 26:654-657.

The online version of this article, along with updated information


and services, is located on the World Wide Web at:
http://www.journalofanimalscience.org/content/26/3/654

www.asas.org

Downloaded from www.journalofanimalscience.org by guest on November 2, 2014

GLYCOLYTIC P A T H W A Y I N R U M E N
DWAYNE

HAIVfAR

MICROORGANISMS 1

2 AND R A Y M O N D B O R C H E R S

University o/ Nebraska, Lincoln


metabolism of carbohydrates ingested
T HE
by ruminants to volatile fatty acids is a
major reaction sequence of rumen microorganisms. These volatile fatty acids are believed
to arise from carbohydrates by the pathway
of glycolysis. The following investigations
were undertaken to determine if glycolysis is
functional in the rumen for the metabolism of
carbohydrates to pyruvate which could then
be converted to volatile fatty acids (Barnett
and Reid, 1961).
Materials and Methods

Rumen material was collected prior to the


morning feeding from fistulated steers on a
maintenance diet of alfalfa and oats and was
strained through four layers of cheesecloth.
Ceil-free extracts (CFE) were prepared from
the freshly strained rumen fluid by centrifuging at 12,000 g for 20 rain. at 4 ~ C. The cells
were washed twice with distilled water and
suspended in a 0.04M potassium maleate
buffer, pH 6.7. The cells were disintegrated
by sonification and the unbroken cells and
debris were removed by centrifuging at
10,000 g for 20 rain. at 4 ~ C.
Glucose was determined by the Folin and
Wu (1919) method or by a modification of the
Huggett and Dixon (1957) method using glucose oxidase. Fructose-6-phosphate was determined by the Roe (1934) method for fructose. Inorganic phosphate was determined by
the Fiske and Subbarow method as modified
by Gomori (1942). Pyruvate was measured
by the decrease in absorption of NADH._, in
the presence of lactate dehydrogenase (Meister, 1950). Two milliliters of a 0.1M sodium
phosphate buffer pH 7.4 containing 3 t*g. of
crystalline enzyme and 150 ~g. NADH_o were
mixed with 1 ml. of the pyruvate sample in a
1 cm. cuvette at room temperature. Immediately thereafter, the decrease in absorption at

340 m~. was followed for 3 rain. with an attached recorder. Aldolase activity was measured by the method of Sibley and Lehninger
(1949) using 2,4-dinitrophenylhydrazine.
Glucose-6-phosphate and glucose were identified by paper chromatography using paper
washed in 2N acetic acid and the following
solvents: ethylene glycol monomethyl ether:
2-butanone: 3M ammonium hydroxide (7 : 2 :
3), acetone: 25% trichloroacetic acid (3:1),
and 95% ehanoh 1M ammonium acetate pH
7.5 (7:3). Pyruvate 2,4-dinitrophenylhydrazone was identified using paper chromatography in n-butanoh 95% ethanol: 0.5M ammonium hydroxide (7: 1:2). Glucose was
detected by an aniline oxalate spray (Block et
al., 1955) and by the use of glucose oxidase
(White and Secor, 1957 ). Glucose-6-phosphate
was detected by an ammonium molybdate
spray (Bandurski and Axelrod, 1951). Pyruvate 2,4-dinitrophenylhydrazone was detected
by spraying with 2% potassium hydroxide in
alcohol (Block et al., 1955).
Barium salts of the sugar phosphates were
dissolved in 0.1N hydrochloric acid and the
barium removed by adding a 10% excess of
sodium sulfate. The barium sulfate was removed by centrifugation and the supernatant
was neutralized with sodium hydroxide. The
concentrations of these solutions are expressed
as percent of the barium salt.
Results

The metabolism of glucose by rumen microorganisms in freshly strained rumen fluid, as


measured by glucose disappearance, was inhibited by iodoacetate at lmM, 0.1ram and
0.01mM to the extent of 84-100%, 50-70%
and 0%, respectively. Fluoride also inhibited
glucose metabolism, 100%, 82-90%, 63-94%
and 50% at 200raM, 100mM, 50mM and
20mM fluoride, respectively.
Glucose-6-phosphate was formed from glucose in a system consisting of CFE, glucose,
1 Published with the approval of the Director as Paper No.
1984, Journal Series, Nebraska Agricultural Experiment StaATP and magnesium sulfate as shown in figtion. Project 15-10 of the Department of Biochemistry and
ure 1. In addition, glucose as determined by
Nutrition contributing to Regional Research Project NC 63.
Some of these data were taken from a thesis submitted by the
glucose oxidase decreased with time. Glucosesenior author to the Graduate College, University of Nebraska,
in partial fulfillment of the requirements for the Ph.D. degree.
6-phosphate formation and glucose disappear2 Present address: Department of Pathology, College of Vetance were dependent upon both glucose and
erinary Medicine, Colorado State University, Fort Collins.
654

Downloaded from www.journalofanimalscience.org by guest on November 2, 2014

R U M E N MICROORGANISMS

655

Figure 1. Formation of glucose-6-phosphate by cell-free extracts of rumen microorganisms. Incubation at 40 ~ of 70 rag. of glucose, 230 rag. of A T P and 25 rag. of magnesium sulfate in 0.7
ml. solution pH 6.7 plus 2.7 ml. of CFE. Chromatogram developed in ethylene glycol monomethyl ether: 2-butanone: 3M ammonium hydroxide (7:2:3) and sprayed with aniline oxalate.

ATP since, when either was omitted from the


system, glucose-6-phosphate could not be detected chromatographically and the glucose
concentration remained constant. Glucose-6phosphate was further shown to be the substance formed by eluting the glucose-6-phosphate from preparative chromatograms and
rechromatographing with two solvents (acetone: 25% trichloroacetic acid and 95% ethanol: 1M ammonium acetate). The same R~
as reference glucose-6-phosphate was observed
in each case. In addition, glucose-6-phosphate
isolated from the reaction mixture by barium
fractionation (Umbreit et at., 1949) was resistant to acid hydrolysis ( I N hydrochloric
acid at 100 ~ for 15 rain.) but was hydrolyzed
by acid phosphatase. Glucose was shown to

be the carbohydrate moiety by detection of


glucose after hydrolysis by glucose oxidase on
paper chromatograms.
The glucose-6-phosphate isomerase conversion of glucose-6-phosphate to fructose-6phosphate by CFE of rumen microorganisms
was demonstrated by measuring (Roe, 1934)
the fructose-6-phosphate formed from glucose6-phosphate. The reaction reached equilibrium in 2 min. with approximately 0.6/zmoles
of fructose-6-phosphate formed from a reaction mixture that consisted of 0.05 ml. of 2 %
glucose-6-phosphate, 0.35 ml. of maleate buffer and 0.1 ml. of CFE.
Ald01ase activity was demonstrated in CFE
of rumen microorganisms, figure 2. Since 2,4dinitrophenylhydrazine could react with fruc-

Downloaded from www.journalofanimalscience.org by guest on November 2, 2014

656

H A M A R AND BORCHERS

6 0.4~

FN
I--

FL
IOmMIOR,0-- -"

0.2

uJ
.~

o.!

_J
0

"~

I0

MINUTES

15

Figure 2. Aldolase activity of cell-free extracts


rumen microorganisms. Three milliliters
of 0.56M hydrazine sulfate pH 6.7, 3 ml. of 4%
fructose diphosphate and 24 ml. of CFE incubated at 40 ~. Activity recorded as the increase
in optical density from 1 ml. of trichloracetic
acid filtrate in the aldolase assay of Sibley and
Lehninger (1949.)
of

tose and fructose-6-phosphate formed as a


result of phosphatase dephosphorylation of
fructose diphosphate, inorganic phosphate was
determined during the measurement of aldolase activity. Inorganic phosphate was found
to increase during the measurement of aldolase
activity. However, 10mM fluoride completely
inhibited phosphate increase but had no
aldolase activity. This clearly indicated that

a: 6 .x i0 2
uJ
-v"
.-I

"'4
o
=

from PG A

=t=L / x ~ , ' f r o m
"' 2
I-.

FDP

,r162
r162

o. 0

15

50
45
MINUTES

60

Figure 3. Pyruvate formation by cell-free extracts of rumen microorganisms. Reaction mixture incubated at 40 ~ for PGA (phosphoglyceric
acid) : 50 /~moles of PGA and 25 mg. manganese
chloride in 5 ml. of 0.04M maleate buffer pH 6.7
plus 20 ml. CFE; and for FDP (fructose diphosphate): 25 mg. of ADP, 50 mg. of NAD
and 25 nag. manganese chloride in 2.5 ml. solution, 2.5 ml. of 4% FDP and 2.5 ml. of 0.1M
sodium dibasic phosphate were mixed and adjusted to pH 6.7 plus 17.5 ml. of CFE.

aldolase activity is present in the CFE of


rumen microorganisms.
Pyruvate was formed from 3-phosphoglycerate or fructose diphosphate as shown in figure
3. The system for the formation of pyruvate
from phosphoglycerate was CFE, phosphoglyceric acid and manganese chloride. The
system for the formation of pyruvate from
fructose diphosphate was CFE, fructose diphosphate, ADP, NAD, manganese chloride
and sodium phosphate. Fluoride completely inhibited the formation of pyruvate from phosphoglycerate at 0.1M fluoride concentration.
Pyruvate was not formed when phosphoglycerate was omitted from the system. Fluoride
completely inhibited the formation of pyruvate from fructose diphosphate at 0.5M concentration; 0.1M iodoacetate resulted in only
30% inhibition. Some pyruvate was formed
when fructose diphosphate was omitted from
the system. Pyruvate was demonstrated to be
a product of the reaction by paper chromatography of the 2,4-dinitrophenylhydrazine
derivative.
Discussion

The metabolism of carbohydrates by rumen


microorganisms in the production of volatile
fatty acids has been studied extensively in regard to the amount of the various volatile
fatty acids formed from different polysaccharities and as a function of dietary conditions. Glycolysis has been felt to be the
pathway of glucose metabolism by rumen microorganisms since lactate could be detected as
an intermediate, and lactate conversion to volatile fatty acids had been demonstrated by
several different authors. Baldwin et al. (1963)
and Pazur et al. (1958) demonstrated with
specifically labeled glucose and xylose, respectively, that the labeling pattern of the acetate
formed from the two carbohydrates agreed
with the labeling pattern which would be expected if glucose and xylose were metabolized
by glycolysis.
The results presented here clearly demonstrate the presence of glucokinase, glucosephosphate isomerase and aldolase in the organisms of the rumen which would be required
for the metabolism of glucose by glycolysis. The conversion of fructose diphosphate
and phosphoglycerate to pyruvate suggests
the presence of glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase and pyruvate kinase. In addition, the results of the fluoride
and iodoacetate inhibition studies support

Downloaded from www.journalofanimalscience.org by guest on November 2, 2014

RUMEN MICROORGANISMS
glycolysis as the pathway of glucose metabolism in the rumen. These results combined
with the isotope studies of Baldwin e t a l .
(1963) clearly indicate that glucose is metabolized to pyruvate b y glycolysis. The pyruvate
formed from glucose by glycolysis could then
be converted to volatile fatty acids by the
pathways reviewed by Barnett and Reid
(1961). I n addition~ pentoses could be metabolized b y glycolysis if the pentoses were converted to hexoses by transaldolase and transketolase reactions.
Palmquist and Baldwin (1966) reported a
change in aldolase activity of cell-free extracts
of rumen microorganisms as a function of diet.
Their results indicated a higher aldolase activity when the animals were fed a concentrate
diet vs. a hay ration. I t would be of interest
to determine if other enzymes of the glycolytic
pathway have a similar change in activity as
a function of diet.
Summary
Glucose metabolism b y rumen microorganisms was inhibited b y fluoride and iodoacetate.
Glucokinase, glucosephosphate isomerase and
aldolase activities were demonstrated b y specific enzyme assays in cell-free extracts of
tureen microorganisms. P y r u v a t e formation
from fructose diphosphate or phosphoglycerate
was demonstrated in incubations with cellfree extracts of rumen microorganisms in the
presence of necessary co-factors.
These results combined with isotope studies
of glucose metabolism clearly indicate that
glucose is metabolized b y glycolysis. T h e pyruvate formed could then be converted to

657

volatile fatty acids as reviewed b y several


authors.
L i t e r a t u r e Cited
Baldwin, R. L., W. A. Wood and R. S. Emery. 1963.
Conversion of glucose-C1. to propionate by the
rumen microbiota. J. Bact. 85:1346.
Bandurski, R. S. and B. Axelrod. 1951. The chromatographic identification of some biologically important phosphate esters. J. Biol. Chem. 193:405.
Barnett, A. J. G. and R. L. Reid. 1961. Reactions in
the Rumen. Edward Arnold, London.
Block, R. J., E. L. Durrum and G. Zweig. 1955.
Paper Chromatography and Paper Electrophoresis.
Academic Press, Inc., New York.
Folin, O. and H. Wu. 1919. A system of blood analysis. J. Biol. Chem. 38:81.
Gomori, G. 1942. A modification of the colorimetric
phosphorus determination for use with the photoelectric colorimeter. J. Lab. Clin. Med. 27:955.
Huggett, A. St. G. and D. A. Dixon. 1957. Enzymic
determination of blood glucose. Biochem. J. 66:12P.
Meister, A. 1950. Reduction of a-diketo and cc-keto
acids catalyzed by muscle preparations and by
crystalline lactic dehydrogenase. J. Biol. Chem.
184:117.
Palmquist, D. L. and R. L. Baldwin. 1966. Enzymatic
techniques for the study of pathways of carbohydrate utilization in the rumen. Appl. Microbiol.
14:60.
Pazur, J. H., E. W. Shuey and C. E. Georgi. 1958.
The conversion of D-xylose into volatile organic
acids by rumen bacteria. Arch. Biochern. Biophys.
77:387.
Roe, J. H. 1934. A colorimetric method for the determination of fructose in blood and urine. J.
Biol. Chem. 107:15.
Sibley, J. A. and A. L. Lehninger. 1949. Determination of aldolase in animal tissue. J. Biol. Chem.
177:859.
Umbreit, W. W., R. H. Burris and ]. F, Stauffer.
1949. Manometric Techniques and Tissue Metabolism. Burgess Publishing Co., Minneapolis.
White, L. M. and G. E. Secor. 1957. Glucose oxidase
with iodide-iodate-starch or o-tolidine as a specific
spray for glucose. Science 125:495.

Downloaded from www.journalofanimalscience.org by guest on November 2, 2014

You might also like