Kinetics of gossypol inhibition of bovine lactate dehydrogenase X

Gossypol, a polyphenolic binaphthalene dialdehyde isolated from cotton meal is a potent inhibitor of lactate dehydrogenase-X purified from bovine testis. For the conversion of pyruvate to lactate the IC50 for gossypol is 200 microM for the reverse reaction the IC50 is 12 microM. Gossypol is a competitive inhibitor of NADH, Ki = 30 microM (Km = 17 microM), and NAD+, Ki = 6 microM (Km = 130 microM), and noncompetitive for pyruvate, Ki = 220 microM (Km = 224 microM), and lactate, Ki = 52 microM (Km = 5.6 mM).     

The half-of-the-sites reactivity of horse liver alcohol dehydrogenase in the presence of alcohol substrates

We investigated by stopped-flow techniques the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase varying the concentration of the reagents, pH and temperature. The course of the reaction under enzymelimiting conditions is biphasic and the measured amplitude of the initial step corresponds under saturation conditions to half of the total enzyme concentration (half-burst). The fast initial step (with a maximum rate of 20 s^?1 at pH 7.0) shows an isotope effect of approximately 2, which indicates that this rate contains a contribution from a hydrogen transfer. It is also shown that this rate differs by at least one order of magnitude with respect to that of the hydrogen transfer during benzaldehyde reduction. The half-of-the-sites reactivity of alcohol dehydrogenase in the initial transient process is obtained independent of reagent concentration, pH and/or temperature. It is obtained also when coenzyme analogues are substituted for NAD, and when different alcohols are substituted for benzyl alcohol. These data are taken to demonstrate unequivocally that the half-of-the-sites reactivity of alcohol dehydrogenase cannot be due to an interplay of rate constants (as proposed by various authors) and must rather be ascribed to a kinetic non-equivalence of the two subunits when active ternary complexes are being formed. When oxidation of benzyl alcohol is carried out in the presence of 0.1 m-isobutyramide (which makes a very tight complex with NADH at the enzyme active site), reaction stops after formation of an amount of NADH product that is equivalent to one half of the enzyme active site concentration.This is considered in the light of the pyrazole experiment designed by McFarland &; Bernhard (1972), in which reduction of benzaldehyde is carried out in the presence of pyrazole (which forms a very tight ternary complex with NAD at the enzyme active site). In this case, reaction stops after formation of an amount of NAD-product which is equivalent to the total enzyme active site concentration. It is shown that accommodation of these two seemingly contradictory sets of data poses severe restrictions on the alcohol dehydrogenase mechanism. In particular, it is shown that the only mechanism that adheres to such requirements is one in which the two subunits have distinct and alternating functions in each enzyme cycle. These two functions are the triggering of the chemical transformation and the chemical transformation itself. It is also shown that binding of NAD-substrate to one subunit triggers chemical reactivity in the other NAD-alcohol-containing subunit, whereas on aldehyde reduction, the triggering event is desorption of alcohol product from the first reacted subunit.     

Reactions of the sulfhydryl groups of alanyl-tRNA synthetase

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PP_i exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn²⁺, which is bound to the enzyme in the absence of ATP.     

Studies of a reactive histidine of dyphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart.

Diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart was inactivated at neutral pH by bromoacetate and diethyl pyrocarbonate and by photooxidation in the presence of methylene blue or rose bengal. Inactivation by diethyl pyrocarbonate was reversed by hydroxylamine. Loss of activity by photooxidation at pH 7.07 was accompanied by progressive destruction of histidine with time; loss of 83% of the enzyme activity was accompanied by modification of 1.1 histidyl residues per enzyme subunit. The pH-rate profiles of inactivation by photooxidation and by diethyl pyrocarbonate modification showed an inflection point around pH 6.6, in accord with the pK_a for a histidyl residue of a protein. Partial protection against inactivation by photooxidation or diethyl pyrocarbonate was obtained with substrate (manganous isocitrate or magnesium isocitrate) or ADP; the combination of substrate and ADP was more effective than the components singly. As demonstrated by differential enzyme activity assays between pH 6.4 and pH 7.5 with and without 0.67 mm ADP, modification of the reactive histidyl residue of the enzyme caused a preferential loss of the positive modulation of activity by ADP. The latter was particularly apparent when substrate partially protected the enzyme against inactivation by rose bengal-induced photooxidation.     

Guanidoacetate methyltransferase from rat liver: Purification,properties, and evidence for the involvement of sulfhydryl groups for activity

Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an α helix content of 15% is calculated. The K_m values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 μm, respectively, at pH 8.0 and 37 °C.     

2-Mercaptoethanol as a substrate for liver alcohol dehydrogenase.

An activity was identified in a phosphate buffer extract of calf liver acetone powder which utilized 2-mercaptoethanol and NAD⁺ as substrates and formed NADH as one product. The activity responsible for catalyzing this reaction is associated with calf liver alcohol dehydrogenase based on copurification, similarity in pH optima, and similarity in response to chelating agents and other inactivating agents. Crystalline horse liver alcohol dehydrogenase also catalyzes the formation of NADH from NAD⁺ using 2-mercaptoethanol as the substrate. Although the K_m for mercaptoethanol is much lower than that for ethanol, 30 μm as compared to 0.625 mm, the maximum velocity with mercaptoethanol as the substrate is only 7% of that when ethanol is the substrate. Because of this difference in maximum velocity, 2-mercaptoethanol is an apparent competitive inhibitor with respect to ethanol with crystalline horse liver alcohol dehydrogenase, consistent with ethanol and 2-mercaptoethanol binding at the same site. The apparent K_i for 2-mercaptoethanol is 14 μm. 2-Butanethiol is a competitive inhibitor with respect to both 2-mercaptoethanol and ethanol with horse and beef liver alcohol dehydrogenases.     

Regulation of glutamate dehydrogenase by palmitoyl-coenzyme A

Glutamate dehydrogenase is inhibited more by palmitoyl-CoA when the reduced form of triphosphopyridine nucleotide instead of the reduced form of diphosphopyridine nucleotide is the coenzyme. Inhibition is further enhanced by α-ketoglutarate and malate. Thus, for example, in the presence of TPNH plus malate, the amount of palmitoyl-CoA required for 50% inhibition is 10-fold lower (0.03 μm) than previously reported values obtained with reduced diphosphopyridine nucleotide as a coenzyme. Allosteric modifiers such as ATP, GTP, and leucine decrease inhibition of glutamate dehydrogenase by palmitoyl-CoA. Palmitoyl-CoA and ADP are competitive. Thus, the palmitoyl-CoA binding site is apparently in the vicinity of the site of these allosteric modifiers and is probably at the ADP site. The fact that ADP (which has only one site per polypeptide chain) can completely prevent inhibition by palmitoyl-CoA suggests that there is only one kinetically significant palmitoyl-CoA binding site per polypeptide chain. This is consistent with the fact that adding one equivalent of palmitoyl-CoA per polypeptide chain inhibits about 80%. The high affinity of glutamate dehydrogenase for palmitoyl-CoA enables it to successfully compete with other mitochondrial proteins for palmitoyl-CoA.     

Electrophoresis of reacting systems involving sulfhydryl oxidation of proteins

Numerical solution of the relevant continuity equations has been used to examine the possible effects of intramolecular sulfhydryl oxidation on the electrophoresis of proteins. Simulations of moving boundary electrophoresis, based on variants of a previous model [J. R. Cann, N. H. Fink, and D. J. Winzor (1983) Arch. Biochem. Biophys.221, 57–63], show that the Schlieren patterns for the ascending and descending limbs are likely to exhibit pronounced nonenantiography. Whereas the pattern for one limb may comprise essentially a single peak, that for the conjugate side can exhibit bimodality, the nature of which is time dependent. Bimodality of the Schlieren pattern can develop in either the ascending or descending limb of the electrophoresis cell, depending basically upon the number of sulfhydryl groups available for oxidation, and on the relative magnitudes of the rate constants describing the oxidation and the isomerization of the oxidized protein species. Whether the faster-moving or slower-moving peak grows with time is shown to depend upon the magnitude of the electrophoretic mobility of the resultant isomer in relation to that of the oxidized protein species. Schlieren patterns for fish muscle creatine kinase and rabbit muscle aldolase are then used to support the relevance of these predictions to moving boundary electrophoresis of proteins undergoing intramolecular sulfhydryl oxidation. Finally, numerical simulation of the zonal electrophoretic behavior of such systems serves to illustrate that bimodal patterns may also obtain, thereby giving a false impression of inherent protein heterogeneity. Emphasis is therefore placed on the importance of maintaining an adequate concentration of reducing agent throughout the medium in which the protein migrates, a potential problem in polyacrylamide gel electrophoresis at neutral pH.     

The environment of the sulfhydryl group in human plasma albumin as determined by spin labeling

A series of spin labels, varying in chain length between the maleimide attaching group and the nitroxide free radical, has been used to investigate the environment of the sulfhydryl group in human plasma albumin. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined and the location of the sulfhydryl was assessed. The effect of bound fatty acids on the sulfhydryl environment was also determined. The environment was found to be analogous to that in the bovine protein, that is, a crevice approximately 9.5 Å deep and not affected in the native state by fatty acids.     

The tryptophan synthase alpha 2 beta 2 complex: a comparison of the reactivity of amino groups in the alpha and beta 2 subunits and in the complex by differential labeling studies

The interaction of the alpha and beta 2 subunits of tryptophan synthase of Escherichia coli to form an alpha 2 beta 2 complex has been probed by differential labeling studies. In the first step the separate alpha or beta 2 subunit or the alpha 2 beta 2 complex was labeled by reductive methylation with trace amounts of [3H]HCHO in the presence of NaCNBH3. In the second step the 3H-labeled preparation was fully labeled under denaturing conditions with [14C]HCHO and NaCNBH3. Peptides containing labeled monomethyl or dimethyl amino groups were isolated after thermolytic digestion or after cyanogen bromide treatment. The 3H/14C ratio of each peptide is a measure of the relative reactivity of the amino group or groups in each peptide. The most reactive amino group in the alpha subunit, lysine-109, is strongly shielded from modification in the alpha 2 beta 2 complex. The most reactive amino group in the beta 2 subunit, the amino-terminal threonine, is not shielded from modification in the alpha 2 beta 2 complex.     

Physical and chemical properties of lactate dehydrogenase in Homarus americanus.

Two isoenzymes of lactate dehydrogenase have been purified from Homarus americanus: One is found predominantly in the tail muscles; the other, in the walking leg muscles. This is the first demonstration of multiple forms of l-specific lactate dehydrogenase in an invertebrate organism. These proteins contain four essential sulfhydryl groups titratable by p-hydroxymercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid). The molecular weights of these isoenzymes are dependent upon ionic strength. The native tetramer (M_r 145,000) exists in low ionic strength solutions; the active dimer (M_r 75,000), in high ionic strength solutions; this is the only example of lactate dehydrogenase disaggregation without concomitant loss in enzymatic activity. Microcomplement fixation studies suggest that there may be less than 4% difference in the primary structures of these two proteins.     

Half-of-the-sites reactivity and all-of-the-sites substrate binding in transaldolase

Transaldolase from Candida utilis is a dimeric protein composed of two identical subunits. The cleavage of fructose 6-phosphate by this enzyme was followed in a rapidmixing spectrophotometer. A very rapid reaction was observed during which 1 mol of glyceraldehyde 3-phosphate/mol of enzyme was released, followed by a much slower reaction in which additional glyceraldehyde 3-phosphate was formed. Binding studies carried out with the same substrate showed that two equivalents of dihydroxyacetone were bound. These results indicate that both sites are active, but that only one functions in the rapid catalytic reaction. The half-of-the-sites reactivity of transaldolase may be attributed to a high degree of negative cooperativity between the two subunits.     

Acute metabolic effects of ammonia on the enzymes of glutamate metabolism in isolated astroglial cells

Enzymes of glutamate metabolism were studied in the astrocytes isolated from rats injected with a large dose of ammonium acetate and compared with those isolated from controls. The activities of glutamate dehydrogenase (GDH) and glutaminase decreased while those of glutamine synthetase (GS) and aspartate aminotransferase (AAT) increased both in convulsive and comatose states. The activity of alanine aminotransferase (A1AT) increased only in convulsive state. The results suggested that glutamate required for the formation of glutamine in astrocytes might have its origin in nerve endings and the depletion of citric acid cycle intermediates might occur in nerve endings at least in acute ammonia toxicity.     

Arachidonate metabolism in bovine gallbladder muscle

Incubation of [1-¹⁴C]arachidonic acid (AA) with homogenates of bovine gallbladder muscle generated a large amount of radioactive material having the chromatographic mobility of 6-keto-PGF_1α (stable product of PGI₂) and smaller amounts of products that comigrated with PGF_2α and PGE₂. Formation of these products was inhibited by the cyclooxygenase inhibitor indomethacin. The major radioactive product identified by thin-layer chromatographic mobility and by gas chromatography - mass spectrometric analysis was found to be 6-keto-PGF_1α. The quantitative metabolic pattern of [1-¹⁴C]PGH₂ was virtually identical to that of [1-¹⁴C]AA. Incubation of arachidonic acid with slices of bovine gallbladder muscle released labile anti-aggregatory material in the medium, which was inhibited by aspirin or 15-hydroperoxy-AA.These results indicate that bovine gallbladder muscle has a considerable enzymatic capacity to produce PGI₂ from arachidonic acid.     

Catalytic properties of lactate dehydrogenase in Homarus americanus.

Lobster tail and leg lactate dehydrogenases (LDH) have been characterized kinetically. The four binding sites for reduced coenzyme have been shown to be equivalent for the enzyme purified from lobster tail muscle. For the reduced form of 3-acetyl pyridineadenine dinucleotide, the K_a = 1.4 × 10⁷ M^?1 S^?1. The activity of the enzyme purified from the tail muscle is severely inhibited (90%) by high levels of pyruvate (10 mm) when assayed for pyruvate reductase activity at 11 °C; the reductase activity measured using the enzyme from the walking leg muscle was not inhibited by these high levels of pyruvate. Evidence is presented which indicates that the LDH from the tail muscle of the East Coast lobster forms an abortive ternary complex (enzyme-NAD⁺-pyruvate) which accounts for these inhibitory kinetics. The data suggest that the LDH from the tail muscles of the invertebrate lobster represents a “kinetic” heart-type l-specific LDH and that from the walking legs, a “kinetic” muscle-type l-specific LDH.     

Kinetics of the inactivation of Escherichia coli glutamate apodecarboxylase by phenylglyoxal.

The possible interaction of the phosphate moiety of pyridoxal phosphate with a guanidinium group in glutamate apodecarboxylase was investigated. The holoenzyme is not inactivated significantly by incubation with butanedione, glyoxal, methylglyoxal, or phenylglyoxal. However, the apoenzyme is inactivated by these arginine reagents in time-dependent processes. Phenylgloxal inactivates the apoenzyme most rapidly. The inactivation follows pseudo-first-order kinetics at high phenylglyoxal to apoenzyme ratios. The rate of inactivation is proportional to phenylglyoxal concentration, increases with increasing pH, and is also dependent on the type of buffer present. The rate of inactivation of the apoenzyme by phenylglyoxal is fastest in bicarbonate — carbonate buffer and increases with increasing bicarbonate — carbonate concentration. Phosphate, which inhibits the binding of pyridoxal phosphate to the apoenzyme, protects the apodecarboxylase against inactivation by phenylglyoxal. When the apodecarboxylase is inactivated with [¹⁴C]phenylglyoxal, approximately 1.6 mol of [¹⁴C]phenylglyoxal is incorporated per mol subunit. The phenylglyoxal is thought to modify an arginyl residue at or near the pyridoxal phosphate binding site of glutamate apodecarboxylase.     

Changes of pyruvate dehydrogenase in brain during alloxan diabetes

Alloxan diabetes causes a decrease in the active form of pyruvate dehydrogenase in rat brain. The effect is severe in the cerebellum and brain stem compared to cerebral hemispheres. The changes observed in the total form are not as significant as those found in the active (dephosphorylated) form. The effects are reversed after administration of insulin to diabetic animals. The severity of diabetes was also found to affect the activity of pyruvate dehydrogenase with inverse correlation. There was a gradual increase in the proportion of active (dephosphorylated) form with increase of time after the onset of diabetes.     

Structural alterations in fibronectin correlated with morphological changes in smooth muscle cells

Nontransformed cultures of vascular smooth muscle cells proliferate until they form a confluent sheet of cells. Subsequently, the cells become reorganized to form multicellular nodules that are loosely attached to the substrate. The formation of nodules is facilitated by the addition of medium conditioned by nodular cultures. Nodulation is inhibited by the addition of fibronectin. Fibronectins derived from monolayer culture conditioned medium or from plasma are maximally effective while fibronectin isolated from nodular cell conditioned medium is inactive. Analysis by NaDodSO4-polyacrylamide gel electrophoresis reveals that the nodular cell fibronectin has a molecular weight that is about 20-30 kd less than that of monolayer cell fibronectin. Further, nodular cell conditioned medium contains an activity that can convert both plasma fibronectin and monolayer cell fibronectin to the lower molecular weight correlated with the loss of biological activity.