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H O Spivey

Publications and source records attributed to H O Spivey.

At least 19 recordsLinked to original sources
PubMed1999

Substrate channeling.

Substrate channeling is the process in which the intermediate produced by one enzyme is transferred to the next enzyme without complete mixing with the bulk phase. This process is equivalent to a microcompartmentation of the intermediate, although classic diffusion occurs simultaneously to varying extents in many of these cases. This microcompartmentation and other factors of channeling provide many potential biological advantages. Extensive examples of channeling can be found in the cited reviews. The choice of methods to detect and characterize substrate channeling depends extensively on the type of enzyme associations involved, the constants of the system, and, to some extent, the mechanism of channeling. Thus it is important to distinguish stable, dynamic, and catalytically induced enzyme associations as well as recognize different mechanisms of substrate channeling. We discuss the principles, experimental details, and limitations and precautions of five rather general methods. These use measurements of transient times, isotope dilution or enhancement, competing reaction effects, enzyme buffering kinetics, and transient-state kinetics. These encompass methods applicable to studies in vitro, in situ, and in vivo. None of these methods is applicable to all systems. They are also susceptible to artifacts without proper attention to precautions. Transient-state kinetic methods clearly excel in elucidating molecular mechanisms of channeling. However, they are often not the best method for initial detection and characterization of the process and they are not applicable to many complex systems. Several other methods that have been successful in indicating substrate channeling are briefly described.

Binding Sites↗
PubMed1999

Detection of kallikrein gene expression and enzymatic activity in porcine endometrium during the estrous cycle and early pregnancy.

Porcine conceptuses rapidly elongate within the uterine horns prior to the period of placental attachment. During the time of elongation, secretion of estrogen by the developing conceptuses occurs for the establishment of pregnancy through maintenance of corpora lutea and facilitation of placental attachment. Factors associated with the uterine luminal epithelium accentuate embryo attachment by allowing close contact between the conceptus and the uterine epithelium. Kallikrein, a serine protease, may be involved with the timing of conceptus expansion and placental attachment to the uterine surface. The objective of this study was to evaluate kallikrein enzymatic activity, protein, and gene expression in the pig during the estrous cycle and early pregnancy. Enzymatic activity was first detected in uterine flushings (UTF) on Day 12 of the estrous cycle and pregnancy. Activity was enhanced on Day 12 of pregnancy compared to that in cyclic gilts, with a reversal of increased kallikrein activity in cyclic compared to pregnant flushings on Day 15. Western blot analysis with antiserum to human plasma kallikrein detected a 50-kDa product similar to human plasma kallikrein from Day 10 to Day 15 of the estrous cycle and pregnancy. Kallikrein enzymatic activity in UTF was associated with the presence of a 23-kDa reactive product. Gene expression of kallikrein as determined by reverse transcription-polymerase chain reaction indicated the presence of kallikrein mRNA in the porcine endometrium and conceptuses. Results indicate that an increase in uterine luminal kallikrein activity occurs during the estrous cycle at a period that corresponds to rapid conceptus elongation during pregnancy of the pig. The present information suggests that kallikrein may play a role in opening the window for establishment of pregnancy in the pig.

Animals↗
PubMed1997

Determination of the specific radioactivity of [14C]lactate by enzymatic decarboxylation and 14CO2 collection.

We present an enzymatic method for the determination of L-[14C]lactate specific radioactivity in complex biological samples containing other radiolabeled compounds. The method is based on the conversion of L-lactate to L-pyruvate by lactate oxidase (no EC number assigned) and the decarboxylation of L-pyruvate by pyruvate oxidase (EC 1.2.3.3). The 14CO2 produced by the enzymatic decarboxylation of pyruvate is quantitatively captured in a CO2 trap and its radioactivity is measured. The method is simple, specific, and precise (2% relative SD). It can be conveniently used for routine multiple determinations of L-[14C]lactate specific radioactivity in tracer metabolic studies. Under specified conditions, the method can also be used to determine the specific radioactivity of L-[14C]pyruvate.

Animals↗
PubMed1994

Binding of malate dehydrogenase and NADH channelling to complex I.

As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochondrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the competing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channelling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction.

Animals↗
PubMed1994

Demonstration of enzyme associations by countermigration electrophoresis in agarose gel.

We propose a method to study multienzyme complex formation in vitro based on nondenaturing agarose gel electrophoresis. The enzymes with different isoelectric points (pI) were loaded at the opposite ends of the same lane of agarose gel and electrophoresis was performed at a pH value intermediate between their pI's. In cases where a complex of the enzymes was formed, an additional protein band of low electrophoretic mobility was found corresponding to the point where they crossed on the gel. This band contained both enzyme activities. The method was used to demonstrate association between two enzymes of the mitochondrial citric acid cycle, malate dehydrogenase and citrate synthase, and between the lysosomal hydrolases, beta-galactosidase and cathepsin A. Relative proportions of free and bound enzymes after electrophoresis suggest that interaction between the mitochondrial enzymes is relatively weak compared to that of lysosomal hydrolases. Microdensitometric scanning of countermigration electrophoresis gels was used to determine the stoichiometry of components in the complex.

Animals↗
PubMed1989

Substrate channeling of NADH and binding of dehydrogenases to complex I.

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1989

Metabolic compartmentation.

Evidence for the association of 'soluble' enzymes in vivo is extensive and compelling. These associations occur in all compartments of the cell of both prokaryotes and eukaryotes. Several factors present in vivo promote these associations among enzymes whose association in vitro is often too weak to detect. Several physiological advantages of the associated enzyme complexes can be identified, most (but not all) of which are the consequence of microcompartmentation of metabolites (substrate channeling). Substrate channeling of intermediates by either a 'direct transfer' process or 'proximity effects' can occur. The latter mechanism does not require the special molecular features needed for the direct transfer mechanism and may, therefore, exist in more general situations in the cell. Criticisms of these views are discussed. We argue that these criticisms have been largely answered by experiment and theory in recent years. Studies on simple systems in vitro, nevertheless, contribute important insights concerning the more complex phenomena in vivo.

Enzymes↗
PubMed1989

Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?

Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D.K. & Bernhard, S.A. (1984) Biochemistry 23, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between alpha-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that NADH is transferred directly between the sites of alpha-GDH and LDH.

Animals↗
PubMed1987

Polyethylene glycol-induced heteroassociation of malate dehydrogenase and citrate synthase.

Studies by dynamic and total intensity light scattering, ultracentrifugation, electron microscopy, and chemical crosslinking on solutions of the pig heart mitochondrial enzymes, malate dehydrogenase and citrate synthase (separately and together) demonstrate that polyethylene glycol induces very large homoassociations of each enzyme, and still larger heteroenzyme complexes between these two enzymes in the solution phase. Specificity of this heteroassociation is indicated by the facts that heteroassociations with bovine serum albumin were not observed for either the mitochondrial dehydrogenase or the synthase or between cytosolic malate dehydrogenase and citrate synthase. The weight fraction of the enzymes in the mitochondrial dehydrogenase-synthase associated particles in the solution phase was less than 0.03% with the dilute conditions used in the dynamic light scattering measurements. Neither palmitoyl-CoA nor other solution conditions tested significantly increased this weight fraction of associated enzymes in the solution phase. Because of the extremely low solubility of the associated species, however, the majority of the enzymes can be precipitated as the heteroenzyme complex. This precipitation is a classical first-order transition in spite of the large particle sizes and broad size distribution. Ionic effects on the solubility of the heteroenzyme complex appear to be of general electrostatic nature. Polyethylene glycol was found to be more potent in precipitating this complex than dextrans, polyvinylpyrrolidones, ficoll, and beta-lactoglobulin.

Animals↗
PubMed1985

Substrate channeling of oxalacetate in solid-state complexes of malate dehydrogenase and citrate synthase.

Current evidence suggests that mitochondrial matrix enzymes exist in solid-state, multienzyme complexes in vivo. Addition of polyethylene glycol to a solution containing malate dehydrogenase and citrate synthase generates such a solid-state, enzyme complex in vitro at enzyme concentrations permitting kinetic measurements. Suspensions of the isolated, solid-state, hetero-complex of these enzymes were used to study the coupled reactions of citrate synthesis from malate, NAD, and CoASAc. The particles appear to be about 1 microgram in diameter. Considering the ratio of enzyme to oxalacetate molecules in or at the surface of the solid-state particles, one would expect oxalacetate to be converted to citrate within a few molecular distances of the site of oxalacetate generation. This model of "substrate channeling" (or alternatively a direct transfer of oxalacetate between enzymes) is supported by experiments with excess aspartate aminotransferase and glutamate added to the solution phase to give a reaction competing with the synthase for bulk phase oxalacetate. Quantities of aminotransferase that reduce the citrate reaction rate with soluble dehydrogenase and synthase by 90% do not significantly affect rates with comparable amounts of the dehydrogenase-synthase complex. We suggest that similar substrate channeling can occur in vivo and discuss the possible advantages provided thereby.

Animals↗
PubMed1979

Ligand-induced conformational transitions and secondary structure composition of chicken liver pyruvate carboxylase.

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO(3) and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10-13%; ATP, 21-24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO(3) had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the epsilon-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499-3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% beta-pleated sheet and 66% other structural types.

Acetyl Coenzyme A↗
PubMed1978

Apparent stability constants of H+ and Mg2" complexes of 5-phosphoribosyl alpha-1-pyrophosphate.

Apparent Mg2+ and H+ stability constants of 5-phosphoribosyl alpha-1-pyrophosphate (ligand, L) complexes were determined from pH titration data at 25 degrees C with an average of 0.17 M NaCl or KCl and 0.20 M ionic strength. The logarithms of calculated macroscopic overall stability constants are: 3.2 (MgL3-), 4.8 (Mg2L-), 6.5 (HL4-), 12.4 H2L3-), 9.4 (Mg HL2-), and 11.0 (MgH2L). Comparison of the stepwise Mg2+ stability constants (log k = 3.2 and 1.6) with those of MgADP- and MgAMP or Mg-hexose-1-P suggests that the first and second Mg2+ bind to the 1-PP and 5-P groups of the ligand, respectively. Reasonable assumptions about relative microscopic constants indicate that several of the microscopic isomers do not achieve significant concentrations over a large range of conditions. Judging from other data on organophosphate complexes, it is likely that the constants of this study may be extrapolated with little error to other conditions of ionic strength 0.1--0.2 M) and temperature (e.g., 15--35 degrees C), and widely different monovalent ion concentrations.

Chemical Phenomena↗
PubMed1978

Hysteretic nature of NADH substrate inhibition of bovine liver glutamate dehydrogenase.

NADH substrate inhibition of bovine liver glutamate dehydrogenase appears to be eliminated at enzyme concentrations above 0.5mg/ml. Since the inhibition cannot be restored by preincubation of the enzyme with any substrate or product combination, the release of inhibition had previously been considered the result of enzyme polymerization. Benzene-saturated solutions, however, increase the extent of enzyme polymerization without affecting the NADH inhibition. These and related control measurements demonstrate that the release of substrate inhibition is the result of a hysteretic transition of an enzyme central and transitory complex.

Animals↗