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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
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
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.
656
H A M A R AND BORCHERS
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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.
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