Dwayne Hamar and Raymond BorchersGlycolytic Pathway in Rumen Microorganisms

1967, 26:654-657.J ANIM SCI

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GLYCOLYTIC PATHWAY IN RUMEN MICROORGANISMS 1

DWAYNE HAIVfAR 2 AND RAYMOND BORCHERS

University o/ Nebraska, Lincoln

T HE metabolism of carbohydrates ingested by ruminants to volatile fatty acids is a major reaction sequence of rumen microor- ganisms. 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 centrifug- ing 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 glu- cose oxidase. Fructose-6-phosphate was deter- mined by the Roe (1934) method for fruc- tose. 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 (Meis- ter, 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. Immedi- ately thereafter, the decrease in absorption at

1 Published with the approval of the Director as Paper No. 1984, Journal Series, Nebraska Agricultural Experiment Sta- tion. Project 15-10 of the Department of Biochemistry and Nutrition contributing to Regional Research Project NC 63. Some of these data were taken from a thesis submitted by the senior author to the Graduate College, University of Nebraska, in partial fulfillment of the requirements for the Ph.D. degree.

2 Present address: Department of Pathology, College of Vet- erinary Medicine, Colorado State University, Fort Collins.

340 m~. was followed for 3 rain. with an at- tached recorder. Aldolase activity was meas- ured by the method of Sibley and Lehninger (1949) using 2,4-dinitrophenylhydrazine.

Glucose-6-phosphate and glucose were iden- tified 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-dinitrophenylhydra- zone was identified using paper chromatogra- phy in n-butanoh 95% ethanol: 0.5M am- monium 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). Pyru- vate 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 re- moved 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 micro- organisms in freshly strained rumen fluid, as measured by glucose disappearance, was in- hibited 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 glu- cose in a system consisting of CFE, glucose, ATP and magnesium sulfate as shown in fig- ure 1. In addition, glucose as determined by glucose oxidase decreased with time. Glucose- 6-phosphate formation and glucose disappear- ance were dependent upon both glucose and

654

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RUMEN 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 ATP 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 ( IN 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 HAMAR AND BORCHERS

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F N I - - o 0.2 uJ

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Figure 2. Aldolase activity of cell-free extracts of rumen microorganisms. Three milliliters of 0.56M hydrazine sulfate pH 6.7, 3 ml. of 4% fructose diphosphate and 24 ml. of CFE in- cubated 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.)

tose and fructose-6-phosphate formed as a result of phosphatase dephosphorylation of fructose diphosphate, inorganic phosphate was determined during the measurement of aldo- lase 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: 2 uJ 6 .x i0 -v" from PG A .-I

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Figure 3. Pyruvate formation by cell-free ex- tracts of rumen microorganisms. Reaction mix- ture 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 di- phosphate): 25 mg. of ADP, 50 mg. of NAD and 25 nag. manganese chloride in 2.5 ml. solu- tion, 2.5 ml. of 4% FDP and 2.5 ml. of 0.1M sodium dibasic phosphate were mixed and ad- justed 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-phosphoglycer- ate or fructose diphosphate as shown in figure 3. The system for the formation of pyruvate from phosphoglycerate was CFE, phospho- glyceric acid and manganese chloride. The system for the formation of pyruvate from fructose diphosphate was CFE, fructose di- phosphate, ADP, NAD, manganese chloride and sodium phosphate. Fluoride completely in- hibited the formation of pyruvate from phos- phoglycerate at 0.1M fluoride concentration. Pyruvate was not formed when phosphogly- cerate was omitted from the system. Fluoride completely inhibited the formation of pyru- vate from fructose diphosphate at 0.5M con- centration; 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 chroma- tography 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 re- gard to the amount of the various volatile fatty acids formed from different polysac- charities and as a function of dietary condi- tions. Glycolysis has been felt to be the pathway of glucose metabolism by rumen mi- croorganisms since lactate could be detected as an intermediate, and lactate conversion to vol- atile 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, respec- tively, that the labeling pattern of the acetate formed from the two carbohydrates agreed with the labeling pattern which would be ex- pected if glucose and xylose were metabolized by glycolysis.

The results presented here clearly demon- strate the presence of glucokinase, glucose- phosphate isomerase and aldolase in the organ- isms of the rumen which would be required for the metabolism of glucose by glyco- lysis. The conversion of fructose diphosphate and phosphoglycerate to pyruvate suggests the presence of glyceraldehydephosphate de- hydrogenase, phosphoglycerate kinase, phos- phoglyceromutase, enolase and pyruvate ki- nase. In addition, the results of the fluoride 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 eta l . (1963) clearly indicate that glucose is metab- olized to pyruvate by 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). In addition~ pentoses could be metab- olized by 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 by rumen microorgan- isms was inhibited by fluoride and iodoacetate. Glucokinase, glucosephosphate isomerase and aldolase activities were demonstrated by spe- cific enzyme assays in cell-free extracts of tureen microorganisms. Pyruvate 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 by glycolysis. The py- ruvate formed could then be converted to

657

volatile fatty acids as reviewed by several authors.

L i te ra ture Cited

Baldwin, R. L., W. A. Wood and R. S. Emery. 1963. Conversion of glucose-C 1. to propionate by the rumen microbiota. J. Bact. 85:1346.

Bandurski, R. S. and B. Axelrod. 1951. The chroma- tographic identification of some biologically impor- tant 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 anal- ysis. J. Biol. Chem. 38:81.

Gomori, G. 1942. A modification of the colorimetric phosphorus determination for use with the photo- electric 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 carbohy- drate 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 de- termination of fructose in blood and urine. J. Biol. Chem. 107:15.

Sibley, J. A. and A. L. Lehninger. 1949. Determina- tion of aldolase in animal tissue. J. Biol. Chem. 177:859.

Umbreit, W. W., R. H. Burris and ]. F, Stauffer. 1949. Manometric Techniques and Tissue Metab- olism. 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.

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