Glycolytic Pathway in Rumen Microorganisms

Dwayne Hamar and Raymond Borchers

J Anim Sci 1967. 26:654-657.

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 GLYCOLYTIC P A T H W A Y I N R U M E N MICROORGANISMS 1
DWAYNE HAIVfAR 2 AND R A Y M O N D B O R C H E R S
University o/ Nebraska, Lincoln

T HE metabolism of carbohydrates ingested

by ruminants to volatile fatty acids is a
340 m~. was followed for 3 rain. with an at-
tached recorder. Aldolase activity was meas-
major reaction sequence of rumen microor- ured by the method of Sibley and Lehninger
ganisms. These volatile fatty acids are believed (1949) using 2,4-dinitrophenylhydrazine.
to arise from carbohydrates by the pathway Glucose-6-phosphate and glucose were iden-
of glycolysis. The following investigations tified by paper chromatography using paper
were undertaken to determine if glycolysis is washed in 2N acetic acid and the following
functional in the rumen for the metabolism of solvents: ethylene glycol monomethyl ether:
carbohydrates to pyruvate which could then 2-butanone: 3M ammonium hydroxide (7 : 2 :
be converted to volatile fatty acids (Barnett 3), acetone: 25% trichloroacetic acid (3:1),
and Reid, 1961). and 95% ehanoh 1M ammonium acetate pH
7.5 (7:3). Pyruvate 2,4-dinitrophenylhydra-
Materials and Methods zone was identified using paper chromatogra-
phy in n-butanoh 95% ethanol: 0.5M am-
Rumen material was collected prior to the monium hydroxide (7: 1:2). Glucose was
morning feeding from fistulated steers on a detected by an aniline oxalate spray (Block et
maintenance diet of alfalfa and oats and was al., 1955) and by the use of glucose oxidase
strained through four layers of cheesecloth. (White and Secor, 1957 ). Glucose-6-phosphate
Ceil-free extracts (CFE) were prepared from was detected by an ammonium molybdate
the freshly strained rumen fluid by centrifug- spray (Bandurski and Axelrod, 1951). Pyru-
ing at 12,000 g for 20 rain. at 4 ~ C. The cells vate 2,4-dinitrophenylhydrazone was detected
were washed twice with distilled water and by spraying with 2% potassium hydroxide in
suspended in a 0.04M potassium maleate alcohol (Block et al., 1955).
buffer, pH 6.7. The cells were disintegrated Barium salts of the sugar phosphates were
by sonification and the unbroken cells and dissolved in 0.1N hydrochloric acid and the
debris were removed by centrifuging at barium removed by adding a 10% excess of
10,000 g for 20 rain. at 4 ~ C. sodium sulfate. The barium sulfate was re-
Glucose was determined by the Folin and moved by centrifugation and the supernatant
Wu (1919) method or by a modification of the was neutralized with sodium hydroxide. The
Huggett and Dixon (1957) method using glu- concentrations of these solutions are expressed
cose oxidase. Fructose-6-phosphate was deter- as percent of the barium salt.
mined by the Roe (1934) method for fruc-
tose. Inorganic phosphate was determined by Results
the Fiske and Subbarow method as modified
by Gomori (1942). Pyruvate was measured The metabolism of glucose by rumen micro-
by the decrease in absorption of NADH._, in organisms in freshly strained rumen fluid, as
the presence of lactate dehydrogenase (Meis- measured by glucose disappearance, was in-
ter, 1950). Two milliliters of a 0.1M sodium hibited by iodoacetate at lmM, 0.1ram and
phosphate buffer pH 7.4 containing 3 t*g. of 0.01mM to the extent of 84-100%, 50-70%
crystalline enzyme and 150 ~g. NADH_o were and 0%, respectively. Fluoride also inhibited
mixed with 1 ml. of the pyruvate sample in a glucose metabolism, 100%, 82-90%, 63-94%
1 cm. cuvette at room temperature. Immedi- and 50% at 200raM, 100mM, 50mM and
ately thereafter, the decrease in absorption at 20mM fluoride, respectively.
Glucose-6-phosphate was formed from glu-
1 Published with the approval of the Director as Paper No. cose in a system consisting of CFE, glucose,
1984, Journal Series, Nebraska Agricultural Experiment Sta- ATP and magnesium sulfate as shown in fig-
tion. Project 15-10 of the Department of Biochemistry and
Nutrition contributing to Regional Research Project NC 63. ure 1. In addition, glucose as determined by
Some of these data were taken from a thesis submitted by the
senior author to the Graduate College, University of Nebraska, glucose oxidase decreased with time. Glucose-
in partial fulfillment of the requirements for the Ph.D. degree.
2 Present address: Department of Pathology, College of Vet-
6-phosphate formation and glucose disappear-
erinary Medicine, Colorado State University, Fort Collins. ance were dependent upon both glucose and
654

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 R U M E N MICROORGANISMS 655

Figure 1. Formation of glucose-6-phosphate by cell-free extracts of rumen microorganisms. In-

cubation 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 mono-
methyl 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 de-
tected chromatographically and the glucose
concentration remained constant. Glucose-6-
phosphate was further shown to be the sub-
stance formed by eluting the glucose-6-phos-
phate from preparative chromatograms and
rechromatographing with two solvents (ace-
tone: 25% trichloroacetic acid and 95% eth-
anol: 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 re-
sistant 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 conver-
sion of glucose-6-phosphate to fructose-6-
phosphate by CFE of rumen microorganisms
was demonstrated by measuring (Roe, 1934)
the fructose-6-phosphate formed from glucose-
6-phosphate. The reaction reached equilib-
rium in 2 min. with approximately 0.6/zmoles
of fructose-6-phosphate formed from a reac-
tion mixture that consisted of 0.05 ml. of 2 %
glucose-6-phosphate, 0.35 ml. of maleate buf-
fer and 0.1 ml. of CFE.
Ald01ase activity was demonstrated in CFE
of rumen microorganisms, figure 2. Since 2,4-
dinitrophenylhydrazine could react with fruc-

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656 H A M A R AND BORCHERS

r aldolase activity is present in the CFE of

6 0.4~ rumen microorganisms.
Pyruvate was formed from 3-phosphoglycer-
ate or fructose diphosphate as shown in figure
FN 3. The system for the formation of pyruvate
from phosphoglycerate was CFE, phospho-
I--
o 0.2 FL
IOmMIOR,0-- -" glyceric acid and manganese chloride. The
system for the formation of pyruvate from
uJ fructose diphosphate was CFE, fructose di-
.~ o.! phosphate, ADP, NAD, manganese chloride
_J
0 and sodium phosphate. Fluoride completely in-
J 0 - ! # hibited the formation of pyruvate from phos-
"~ 0 5 I0 15 phoglycerate at 0.1M fluoride concentration.
MINUTES Pyruvate was not formed when phosphogly-
Figure 2. Aldolase activity of cell-free extracts cerate was omitted from the system. Fluoride
of rumen microorganisms. Three milliliters completely inhibited the formation of pyru-
of 0.56M hydrazine sulfate pH 6.7, 3 ml. of 4% vate from fructose diphosphate at 0.5M con-
fructose diphosphate and 24 ml. of CFE in- centration; 0.1M iodoacetate resulted in only
cubated at 40 ~. Activity recorded as the increase
in optical density from 1 ml. of trichloracetic 30% inhibition. Some pyruvate was formed
acid filtrate in the aldolase assay of Sibley and when fructose diphosphate was omitted from
Lehninger (1949.) the system. Pyruvate was demonstrated to be
a product of the reaction by paper chroma-
tose and fructose-6-phosphate formed as a tography of the 2,4-dinitrophenylhydrazine
result of phosphatase dephosphorylation of derivative.
fructose diphosphate, inorganic phosphate was
determined during the measurement of aldo- Discussion
lase activity. Inorganic phosphate was found The metabolism of carbohydrates by rumen
to increase during the measurement of aldolase microorganisms in the production of volatile
activity. However, 10mM fluoride completely fatty acids has been studied extensively in re-
inhibited phosphate increase but had no gard to the amount of the various volatile
aldolase activity. This clearly indicated that fatty acids formed from different polysac-
charities and as a function of dietary condi-
tions. Glycolysis has been felt to be the
a: 6 .x i0 2
uJ pathway of glucose metabolism by rumen mi-
-v" from PG A croorganisms since lactate could be detected as
.-I an intermediate, and lactate conversion to vol-
atile fatty acids had been demonstrated by
"'4
=
o / several different authors. Baldwin et al. (1963)
and Pazur et al. (1958) demonstrated with
=t=L / x ~ , ' f r o m FDP specifically labeled glucose and xylose, respec-
tively, that the labeling pattern of the acetate
"' 2 formed from the two carbohydrates agreed
I-.
,r162 with the labeling pattern which would be ex-
pected if glucose and xylose were metabolized
r162 by glycolysis.
The results presented here clearly demon-
o. 0 15 50 45 60 strate the presence of glucokinase, glucose-
MINUTES phosphate isomerase and aldolase in the organ-
Figure 3. Pyruvate formation by cell-free ex- isms of the rumen which would be required
tracts of rumen microorganisms. Reaction mix- for the metabolism of glucose by glyco-
ture incubated at 40 ~ for PGA (phosphoglyceric lysis. The conversion of fructose diphosphate
acid) : 50 /~moles of PGA and 25 mg. manganese
chloride in 5 ml. of 0.04M maleate buffer pH 6.7 and phosphoglycerate to pyruvate suggests
plus 20 ml. CFE; and for FDP (fructose di- the presence of glyceraldehydephosphate de-
phosphate): 25 mg. of ADP, 50 mg. of NAD hydrogenase, phosphoglycerate kinase, phos-
and 25 nag. manganese chloride in 2.5 ml. solu- phoglyceromutase, enolase and pyruvate ki-
tion, 2.5 ml. of 4% FDP and 2.5 ml. of 0.1M
sodium dibasic phosphate were mixed and ad- nase. In addition, the results of the fluoride
justed to pH 6.7 plus 17.5 ml. of CFE. and iodoacetate inhibition studies support

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 RUMEN MICROORGANISMS
glycolysis as the pathway of glucose metab-
olism in the rumen. These results combined
with the isotope studies of Baldwin e t a l .
(1963) clearly indicate that glucose is metab-
olized 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 metab-
olized b y glycolysis if the pentoses were con-
verted to hexoses by transaldolase and trans-
ketolase 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 activ-
ity 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 microorgan-
isms was inhibited b y fluoride and iodoacetate.
Glucokinase, glucosephosphate isomerase and
aldolase activities were demonstrated b y spe-
cific 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 cell-
free 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 py-
ruvate formed could then be converted to
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657
volatile fatty acids as reviewed b y several
authors.
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