ENZYMES OF PYRUVATE AND PHOSPHOENOLPYRUVATE CARBOXYLATION IN RUMEN BACTERIA

1974 ◽  
Vol 54 (4) ◽  
pp. 595-603 ◽  
Author(s):  
A. S. ATWAL ◽  
F. D. SAUER
Keyword(s):  
Calcium Phosphate ◽  
Ammonium Sulphate ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Rumen Fluid ◽  
Maximum Activity ◽  
Rumen Bacteria ◽  
Carboxylase Activity ◽  
Mixed Bacteria ◽  
Pep Carboxykinase

Extracts of mixed bacteria collected from bovine rumen fluid contained enzymes that carboxylated pyruvate and phosphoenolpyruvate (PEP). Fresh extracts showed high pyruvate carboxylase activity (EC 6.4.1.1. pyruvate carboxylase) which, however, was cold-labile and lost activity on dialysis at 4 C. Enzyme(s) catalyzing the carboxylation of PEP were stable under these conditions. The carboxylation of PEP was maximally stimulated by ADP and to a lesser degree by GDP. The ADP–requiring PEP carboxykinase [EC 4.1.1.49 phosphoenolpyruvate carboxykinase (ATP)] was equally active with either Mg++ or Mn++ and showed maximum activity at pH 6.5. The GDP–requiring PEP carboxykinase [EC 4.1.1.32 phosphoenolpyruvate carboxykinase (GTP)] required Mn++ and was almost inactive if Mg++ was substituted. Maximum activity was at pH 7.0. These nucleotides were most effective at 2.5-mM concentration and were inhibitory at higher concentrations. In the absence of added ADP or GDP, the carboxylation of PEP continued at a low but persistent rate. Precipitation with ammonium sulphate and adsorption on calcium phosphate gel resulted in fractions containing different proportions of the three activities. These results suggest that in mixed rumen bacterial extracts there are four separate enzymes capable of synthesizing oxaloacetate: one that catalyzes the carboxylation of pyruvate and three that catalyze the carboxylation of PEP.

Biphasic breakdown of peptides by rumen bacteria

1996 ◽  
Vol 1996 ◽  
pp. 237-237
Author(s):  
R.J. Wallace
Keyword(s):  
Dry Matter ◽  
Rumen Fluid ◽  
Rumen Bacteria ◽  
Mixed Diet ◽  
Supernatant Fluid ◽  
Differential Centrifugation ◽  
Ammonia Production ◽  
Protein Breakdown ◽  
Adult Sheep ◽  
Mixed Bacteria

Excessive protein breakdown and ammonia production often lead to inefficient N utilisation in ruminants. Peptides are intermediates in the breakdown process. The aims of this study were to characterize peptidase activities of rumen bacteria and to determine which species of bacteria were responsible for the different steps of peptide breakdown in the rumen.Four rumen-fistulated adult sheep received a mixed diet (hay, barley, molasses, fishmeal and minerals and vitamins: 500, 99.5, 100, 91 and 9.5 g/kg dry matter respectively) fed in equal meals of 500 g at 0800 and 1600 h. Rumen fluid was removed 3 h after the morning feeding, strained, and mixed bacteria were prepared by differential centrifugation. Bacteria were sonicated and debris was removed by centrifugation. Peptidase activities were determined on the supernatant fluid.

scholarly journals The Pyruvate Carboxylase of Verticillium albo-atrum

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 15-19 ◽  
Author(s):  
R. E. Hartman ◽  
N. T. Keen
Keyword(s):  
Pyruvate Carboxylase ◽  
Krebs Cycle ◽  
Physiological Role ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Carboxylase Activity ◽  
Metabolic Intermediates ◽  
Krebs Cycle Intermediates ◽  
Verticillium Albo Atrum

The pyruvate carboxylase of Verticillium albo-atrum had a pH optimum of 7·8 and a specific requirement for ATP. At the optimum pH, magnesium ions were required for maximum activity, while at pH 6·8 manganese was more effective than magnesium. Potassium was stimulatory while sodium was ineffective. Avidin and p-chloromercuribenzoate strongly inhibited the enzyme while biotin and dithiothreitol, respectively, reversed the effect of the inhibitors. Aspartate and oxalacetate were inhibitory while acetyl-CoA and CoA reversed the inhibition by aspartate. These cofactors were ineffective in the absence of aspartate. None of the tested metabolic intermediates was stimulatory to pyruvate carboxylase activity while NADP+ and 2,3-diphosphoglycerate were the most effective inhibitors (75%) at a concentration of 6·7 mM. Levels of pyruvate carboxylase in cells grown on glucose, acetate, malate, xylose, glycerol or aspartate differed only slightly. The data indicated that the physiological role of pyruvate carboxylase in V. albo-atrum is the anaplerotic biosynthesis of C4 Krebs-cycle intermediates from pyruvate.

scholarly journals The activities and intracellular distribution of nicotinamide-adenine dinucleotide phosphate-malate dehydrogenase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in rat, guinea-pig and rabbit tissues

1975 ◽  
Vol 146 (2) ◽  
pp. 329-332 ◽  
Author(s):  
D E Saggerson ◽  
C J Evans
Keyword(s):  
Adipose Tissue ◽  
Guinea Pig ◽  
Malate Dehydrogenase ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Intracellular Distribution ◽  
Kidney Cortex ◽  
Carboxylase Activity ◽  
Liver And Kidney

1. Measurements are presented of the activity and intracellular distribution of phosphoenolypruvate carboxykinase, pyruvate carboxylase and NADP-malate dehydrogenase in rat, guinea-pig and rabbit liver and kidney cortex, together with previously obtained measurements of these enzymes in adipose tissue. 2. In all three tissues pyruvate carboxylase activity was greatest in the rat and lowest in the rabbit. 3. Guinea pig and rabbit were very similar to each other with respect to the extramitochondrial-mitochondrial distribution of phosphoenolpyruvate carboxykinase in all three tissues. 4. NADP-malate dehydrogenase was present in all three tissues in the rat, present in kidney cortex and adipose tissue in the guinea pig and absent from all tissues examines in the rabbit.

scholarly journals Digestion of rumen bacteria in vitro

1983 ◽  
Vol 49 (1) ◽  
pp. 101-108 ◽  
Author(s):  
R. J. Wallace
Keyword(s):  
Rumen Fluid ◽  
Individual Species ◽  
Rumen Bacteria ◽  
Gram Negative Bacteria ◽  
Bacterial Protein ◽  
Gram Positive ◽  
Gram Negative ◽  
Pure Cultures ◽  
Mixed Bacteria

1. A pepsin + pancreatin method was used to assess the digestibility of pure cultures of rumen bacteria and mixed bacteria prepared from rumen fluid.2. Individual species of Gram-negative rumen bacteria were highly digestible, whereas Gram-positive species, especially cocci, were more resistant to digestion.3. A similar difference was observed microscopically with mixed rumen bacteria, but the influence of the relative proportions of Gram-positive and Gram-negative bacteria on the digestibility of bacterial protein in rumen fluid was small.

scholarly journals Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats

1987 ◽  
Vol 245 (3) ◽  
pp. 661-668 ◽  
Author(s):  
M Rigoulet ◽  
X M Leverve ◽  
P J A M Plomp ◽  
A J Meijer
Keyword(s):  
Steady State ◽  
Production Rate ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glucose Production ◽  
Carboxylase Activity ◽  
Maximal Stimulation ◽  
Intracellular Concentrations ◽  
Effect Of Glucose ◽  
Stimulation Of

Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity.

Activation of phosphoenolpyruvate carboxykinase isolated from Veillonella parvula

1986 ◽  
Vol 64 (9) ◽  
pp. 898-905
Author(s):  
Helen S. Chau ◽  
Stephen K. Ng
Keyword(s):  
Ammonium Sulphate ◽  
Column Chromatography ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Maximum Activity ◽  
Acetyl Phosphate ◽  
Acetyl Coa ◽  
Protamine Sulphate ◽  
Ammonium Sulphate Precipitation ◽  
Fructose 1,6 Bisphosphate

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) has been purified 940-fold from Veillonella parvula using protamine sulphate treatment, ammonium sulphate precipitation, and column chromatography. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity in the direction of oxaloacetate (OAA) decarboxylation was exhibited at pH 9.0. At this pH, the V. parvula enzyme catalysed phosphenolpyruvate formation in the presence of Mn2+ ions. In the presence of varying concentrations of OAA and ATP, the PEPCK from V. parvula exhibited hyperbolic kinetics with Kms of 0.16 and 0.46 mM, respectively. PEPCK from the anaerobe was not inhibited by NADH, succinate, glutamate, D-glucose 6-phosphate, acetyl phosphate, D-fructose 1,6-bisphosphate, pyruvate, ribose 5-phosphate, and aspartate. However, acetyl CoA, glyceraldehyde 3-phosphate, 3-phospho-D-glycerate, CTP, and GTP activated the enzyme. The activation of acetyl CoA was uncompetitive and noncooperative.

Acute suppression of hepatic gluconeogenesis by glucose in the intact animal

1975 ◽  
Vol 229 (6) ◽  
pp. 1569-1575 ◽  
Author(s):  
HG McDaniel
Keyword(s):  
Fatty Acid ◽  
Blood Glucose ◽  
Pyruvate Carboxylase ◽  
Octanoic Acid ◽  
Intact Animal ◽  
Liver Glycogen ◽  
Acid Oxidation ◽  
Carboxylase Activity ◽  
Glucose Administration ◽  
Pep Carboxykinase

Within 2 h after glucose administration to fasting rats the incorporation of radioactive lactate into blood glucose and liver glycogen is decreased. Using tryptophan, which facilitates the study of gluconeogenesis prior to the phosphoenolpyruvate (PEP) carboxykinase step by increasing the level of certain hepatic metabolites, we have found that in animals fasted for 24 h glucose markedly decreased hepatic malate and aspartate concentrations without a corresponding fall in that of pyruvate, suggesting a decrease in pyruvate carboxylase activity. An inhibitor of fatty acid oxidation, 4-pentenoic acid, similarly decreased the accumulation of these intermediates, and octanoic acid significantly lessened the fall in malate and aspartate with glucose. The changes in tissue metabolite levels were consistent with inhibition of the liver pyruvate carboxylase reaction by glucose treatment, and with abolition of this inhibition by octanoate administration. Alanine and glutamate levels in the liver of tryptophan-treated animals were decreased 90 and 32%, respectively, by glucose. Thus, glucose administration in the whole animal acutely decreases gluconeogenesis by apparently inhibiting the pyruvate carboxylase step and decreasing alanine levels in the liver.

Biphasic breakdown of peptides by rumen bacteria

1996 ◽  
Vol 1996 ◽  
pp. 237-237
Author(s):  
R.J. Wallace
Keyword(s):  
Dry Matter ◽  
Rumen Fluid ◽  
Rumen Bacteria ◽  
Mixed Diet ◽  
Supernatant Fluid ◽  
Differential Centrifugation ◽  
Ammonia Production ◽  
Protein Breakdown ◽  
Adult Sheep ◽  
Mixed Bacteria

Excessive protein breakdown and ammonia production often lead to inefficient N utilisation in ruminants. Peptides are intermediates in the breakdown process. The aims of this study were to characterize peptidase activities of rumen bacteria and to determine which species of bacteria were responsible for the different steps of peptide breakdown in the rumen.Four rumen-fistulated adult sheep received a mixed diet (hay, barley, molasses, fishmeal and minerals and vitamins: 500, 99.5, 100, 91 and 9.5 g/kg dry matter respectively) fed in equal meals of 500 g at 0800 and 1600 h. Rumen fluid was removed 3 h after the morning feeding, strained, and mixed bacteria were prepared by differential centrifugation. Bacteria were sonicated and debris was removed by centrifugation. Peptidase activities were determined on the supernatant fluid.

scholarly journals Some factors influencing the chemical composition of mixed rumen bacteria

1974 ◽  
Vol 31 (1) ◽  
pp. 27-34 ◽  
Author(s):  
R. H. Smith ◽  
A. B. Mcallan
Keyword(s):  
Chemical Composition ◽  
Dry Matter ◽  
Rumen Bacteria ◽  
Host Animal ◽  
Adult Cattle ◽  
Total N ◽  
Adult Sheep ◽  
Factors Influencing ◽  
Mixed Bacteria ◽  
Ciliate Protozoa

1. Sheep, cows and calves fitted with rumen cannulas were given diets mostly containing 10–16 g nitrogen/kg dry matter and consisting of roughage and cereals. Mixed bacteria were separated from samples of their rumen contents.2. Bacteria taken 4–6 h after a feed from calves which were kept in an experimental calf-house with no contact with adult animals (environment A) contained more α-dextran, less total N and higher nucleic acid:total N ratios than similar bacteria from calves reared in contact with adult sheep (environment C) but otherwise treated in an identical way.3. Mixed bacteria taken 4–6 h after a feed from sheep and cows were similar in composition, with respect to nitrogenous components, to those from the ‘environment C’ calves. This composition did not vary significantly when diets containing differing proportions of roughage were given.4. The ‘environment A’ calves were free of ciliate protozoa. When they were placed in contact with, and were inoculated with rumen contents from, adult cattle (environment B), they rapidly developed a normal protozoal population and the chemical composition of their rumen bacteria became like that of the bacteria from the ‘environment C’ calves.5. Mixed bacteria taken just before a feed, from either cows or ‘environment A’ calves, showed significantly lower RNA-N:total N ratios and slightly (but not usually significantly) higher DNA-N:total N ratios than bacteria taken 4–6 h after feeding. Total N contents of the bacteria did not change consistently with time after feeding.6. The possible significance of these differences in relation to the nutrition of the host animal is discussed.

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