Purification of 3 alpha-hydroxysteroid and 3 beta-hydroxysteroid dehydrogenases from human liver cytosol.

We previously reported that the human Y' bile acid binder, which has higher bile acid binding affinities than rat Y' binders (3 alpha-hydroxysteroid dehydrogenases), has dihydrodiol dehydrogenase activity and is different from 3 alpha-hydroxysteroid dehydrogenases. In this study, 3 alpha-hydroxysteroid dehydrogenases and 3 beta-hydroxysteroid dehydrogenase were purified from human liver, and bile acid binding affinities and enzyme kinetics of the 3 alpha-hydroxysteroid dehydrogenases were studied. On chromatofocusing of pooled Affigel blue fraction of the Y' fraction, three 3 alpha-hydroxysteroid dehydrogenase peaks eluted at pH 6.0, 5.7 and 5.4. These peaks did not bind bile acids, and further purification by hydroxyapatite-high-performance liquid chromatography gave pure 3 alpha-hydroxysteroid dehydrogenases with identical M(r) (36,000) having dihydrodiol dehydrogenase activity. 3 beta-Hydroxysteroid dehydrogenase was eluted together with Y' bile acid binder at pH 7.2 on chromatofocusing and was separated from Y' bile acid binder on hydroxyapatite-high-performance liquid chromatography as a pure protein with M(r) 32,000. The apparent Kms of 3 alpha-hydroxysteroid dehydrogenases were similar to those of rat enzymes. In conclusion, we purified human hepatic 3 alpha-hydroxysteroid dehydrogenases, which have similar characteristics to rat enzymes, but do not bind bile acids or reduce bile acid precursors. These data further support the importance of human bile acid binder in intracellular bile acid transport in the human liver.     

Purification and Separation of Pyridine Nucleotide-Linked Dehydrogenases by Affinity Chromatography Techniques

A number of different dehydrogenases have been shown to bind to Sepharose-bound N(6)-(6-aminohexyl)-AMP. These dehydrogenases can be specifically eluted by binary adducts of NAD(+) or with cofactor gradients. In such manner pure enzymes can be obtained from crude extracts, as demonstrated in the purification on a preparative scale of lactate dehydrogenase from dogfish muscle. The data presented indicate the usefulness of general ligands as affinity agents. The techniques are particularly adaptable for the isolation of human mutant enzymes in blood or in the purification and concentration of enzymes present at low levels in fluids or tissues, as shown in the extensive purification of serum lactate dehydrogenase and glucose 6-phosphate dehydrogenase from hemolysate. lsoenzymes with different affinities for co-enzymes can be separated by affinity techniques. Application of affinity techniques may lead to the separation of isoenzymes or mutant enzymes that are not separable by electrophoretic methods.     

Isovaleryl-CoA dehydrogenase: demonstration in rat liver mitochondria by ion exchange chromatography and isoelectric focusing.

There has been ambiguity concerning the specificity of the enzymes that dehydrogenate short branched-chain acyl-CoAs. It previously had been assumed that isovaleryl-CoA is dehydrogenated by n-butyryl-CoA dehydrogenase [butyryl-CoA:(acceptor) oxidoreductase, EC 1.3.99.2]. To solve this problem, we fractionated five short-chain acyl-CoA dehydrogenases (isovaleryl-CoA, n-butyryl-CoA, isobutyryl-CoA, n-octanoyl-CoA, and glutaryl-CoA dehydrogenases) from rat liver mitochondria by isoelectric focusing and DEAE-cellulose column chromatography. The isovaleryl-CoA dehydrogenase [isovaleryl-CoA:(acceptor) oxidoreductase, EC 1.3.99.10] peak was almost completely separated from the peaks of n-butyryl CoA- and n-octanoyl-CoA dehydrogenases by isoelectric focusing, and it was well separated from glutaryl-CoA dehydrogenase [glutaryl-CoA:(acceptor) oxidoreductase (decarboxylating), EC 1.3.99.7] and n-octanoyl-CoA dehydrogenase by DEAE-cellulose column chromatography. The isovaleryl-CoA dehydrogenase peak partly overlapped that of n-butyryl-CoA and isobutyryl-CoA dehydrogenases in the latter procedure. These results unequivocally demonstrate that isovaleryl-CoA is oxidized by a specific isovaleryl-CoA dehydrogenase. The other dehydrogenase peaks also demonstrated activity toward a single substrate, except that isobutyryl-CoA dehydrogenase activity could not be clearly resolved from n-butyryl-CoA dehydrogenase activity.     

Reaction site of carboxanilides and of thenoyltrifluoroacetone in complex II.

Oxathiin carboxanilides are systemic fungicides that inhibit the oxidation of succinate by interrupting electron transport between succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1] and coenzyme Q. Kinetic and electron paramagnetic resonance studies have established that the specific binding site of carboxanilides and of thenoyltrifluoroacetone responsible for the inhibition is the same. Although the binding of carboxanilides to membrane preparations of the dehydrogenase is very tight (Ki = 0.01-0.1 microM), it is noncovalent. Identification of the membrane component(s) to which specific binding occurs has therefore required the introduction of a photoaffinity label onto the carboxanilide molecule. By using [G-3H]3'-azido-5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide, it was found, in accord with earlier data with other carboxanilides, that unresolved complex II specifically binds about 0.6 mol of the inhibitor per mol of succinate dehydrogenase in equilibrium dialysis experiments. The resolved components of the complex, succinate dehydrogenase and the two binding peptides CII-3 and CII-4, failed to bind the inhibitor; however, when these were recombined with reconstitution of coenzyme Q reductase activity, the initial binding titer was restored. Azidocarboxanilide-inhibited complex II was irradiated to generate covalent linkages with the binding site, and the components of the complex were separated on polyacrylamide gel. Most of the specifically bound inhibitor was found in the low molecular weight binding peptides and phospholipids.     

Human Brain Glyceraldehyde-3-Phosphate Dehydrogenase, Succinic Semialdehyde Dehydrogenase and Aldehyde Dehydrogenase Isozymes: Substrate Specificity and Sensitivity to Disulfiram

Several enzymes active in the presence of NAD with acetaldehyde and propionaldehyde have been purified from human brain and characterized. The enzymes have been identified as aldehyde dehydrogenase (EC 1.2.1.3), NAD-linked succinic semialdehyde dehydrogenase (EC 1.2.1.24), and glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). Glyceraldehyde-3-phosphate dehydrogenase is extremely heterogeneous with some isozymes active with acetaldehyde, others inactive. The cytoplasmic enzyme, which is the classical glyceraldehyde-3-phosphate dehydrogenase, is inactive with acetaldehyde as substrate; the isozymes that are active with short chain aliphatic aldehydes are localized in the mitochondrial fraction. Properties of glyceraldehyde-3-phosphate dehydrogenase isozymes with respect to short chain aliphatic aldehydes and inhibition by disulfiram are described. Their Km values for acetaldehyde range from 300 to 2000 microM. All glyceraldehyde-3-phosphate dehydrogenases that are active with acetaldehyde are easily inactivated by low concentrations of disulfiram. In all cases activity regain can be obtained with 2-mercaptoethanol; in the case of two glyceraldehyde-3-phosphate isozymes (E8.5 and 9.0), activity can also be regained with cysteine and with glutathione; activity of E6.6 and E6.8 glyceraldehyde-3-phosphate dehydrogenases could not be regained with 33 microM cysteine or glutathione. Succinic semialdehyde dehydrogenase and aldehyde dehydrogenase (EC 1.2.1.3) were also inhibited by disulfiram; their activity could be regained with 2-mercaptoethanol but not with 33 microM cysteine or glutathione. Comparison of human brain succinic semialdehyde dehydrogenase and aldehyde dehydrogenase with glyceraldehyde-3-phosphate dehydrogenase shows that the activity with short chain aldehydes is not unique to aldehyde dehydrogenase; neither is sensitivity to disulfiram; activity with 3,4-dihydroxyphenylacetaldehyde appears to be a unique property of aldehyde dehydrogenase (EC 1.2.1.3).     

Subcellular targeting analysis of SDR-type hydroxysteroid dehydrogenases

Most mammalian hydroxysteroid dehydrogenases known thus far belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) and aldo-keto reductases (AKR). Whereas members of the AKR family are soluble, cytoplasmic enzymes, SDR-type hydroxysteroid dehydrogenases are also located to other subcellular compartments, i.e. endoplasmic reticulum, mitochondria or peroxisomes. Differential localization might play an important role in influencing the reaction direction of hydroxy dehydrogenase/oxo reductase pathways by determining the available nucleotide cofactor pool. Targeting signals for different subcellular organelles in human hydroxysteroid dehydrogenases have been identified, however, in several enzymes localization signals remain to be determined.     

Alcohol and polyol dehydrogenases are both divided into two protein types, and structural properties cross-relate the different enzyme activities within each type.

Sorbitol dehydrogenase from sheep liver shows similarities to mammalian and yeast alcohol dehydrogenases. Comparisons based on peptides from segments of sorbitol dehydrogenase reveal that homologous regions with 38% identity include two ligands to the active site zinc atom in liver alcohol dehydrogenase, as well as further important residues. Similarities in in other regions are less extensive, exactly as they are between different alcohol dehydrogenases. In all aspects, sorbitol dehydrogenase appears as a typical member of the alcohol dehydrogenase family. On the other hand, alcohol dehydrogenase from Drosophila, which has a shorter subunit, is not closely related to either of these enzymes, except for a region that probably corresponds to the first part of the coenzyme binding domain in many dehydrogenases. Instead, Drosophila alcohol dehydrogenase in its supposed catalytic region shows similarities toward Klebsiella ribitol dehydrogenase, which also has a small subunit. It may be concluded that both alcohol and polyol dehydrogenases show two types of protein subunit, reflecting an early subdivision of polypeptide types into "long" and "short" subunits rather than into different enzymatic specificities or quaternary structures. The relationships explain known properties of all these enzymes and provide insight into functional mechanisms and evolutionary interpretations.     

Structure of L-3-hydroxyacyl-coenzyme A dehydrogenase: preliminary chain tracing at 2.8-A resolution.

The conformation of L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) has been derived from electron-density maps calculated at 2.8-A resolution with phases obtained from two heavy-atom derivatives and the bound coenzyme, NAD. Like other dehydrogenases, 3-hydroxyacyl-CoA dehydrogenase is a double-domain structure, but the bilobal nature of this enzyme is more pronounced than has been previously observed. The amino-terminal domain, which comprises approximately the first 200 residues, is responsible for binding the NAD cofactor and displays considerable structural homology with the dinucleotide binding domains observed in other NAD-, NADP-, and FAD-dependent enzymes. The carboxyl-terminal domain, comprising the remaining 107 residues, appears to be all alpha-helical and bears little homology to other known dehydrogenases. The subunit-subunit interface in the 3-hydroxyacyl-CoA dehydrogenase dimer is formed almost exclusively by residues in the smaller helical domain. A difference map between the apo and holo forms of the crystalline enzyme has been interpreted in terms of the NAD molecule being bound in a typically extended conformation. The location of the coenzyme binding site, along with the structural homology to other dehydrogenases, makes it possible to speculate about the location of the binding site for the fatty acyl-CoA substrate.     

A 15-hydroxyprostaglandin dehydrogenase specific for prostaglandin A in rabbit kidney.

Examination of a soluble fraction derived from homogenates of rabbit kidney papilla revealed the existence of a 15-hydroxyprostaglandin dehydrogenase specific for A-type prostaglandins. Prostaglandins of the E- and F-series were not substrates for this enzyme. In agreement with published data, the 15-hydroxyprostaglandin dehydrogenase(s) derived from the kidney cortex were found to degrade all prostaglandins examined (PGE, PGF, PGA) in the presence of added cofactor NAD. Thus it is evident that in this species the kidney 15-hydroxyprostaglandin dehydrogenases are anatomically compartmentalized so that the papilla is able to metabpable of degrading E-, F-, and A-type prostaglandins by this metabolic pathway.     

Escherichia coli succinate dehydrogenase variant lacking the heme b

The Escherichia coli enzyme succinate:ubiquinone oxidoreductase [(succinate dehydrogenase (SdhCDAB)] couples succinate oxidation to ubiquinone reduction and is structurally and functionally equivalent to mitochondrial complex II, an essential component of the aerobic respiratory chain and tricarboxylic acid cycle. All such enzymes contain a heme within their membrane anchor domain with a highly contentious, but as-yet-undetermined, function. Here, we report the generation of a complex II that lacks heme, which is confirmed by both optical and EPR spectroscopy. Despite the absence of heme, this mutant still assembles properly and retains physiological activity. However, the mutants lacking heme are highly sensitive to the presence of detergent. In addition, the heme does not appear to be involved in reactive oxygen species suppression. Our results indicate that redox cycling of the heme in complex II is not essential for the enzyme's ubiquinol reductase activity.     

Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity.     

Loss of respiratory enzyme activity in anoxic myocardium effect of propranolol

Guinea pig hearts were perfused anoxically with and without dl-propranolol hydrochloride (0.1 mg.l⁻¹) to determine whether a therapeutic level of this drug could protect the inner mitochondrial membrane. After 5 h anoxia, the specific activities of the mitochondrial inner membrane enzymes, succinate dehydrogenase and cytochrome oxidase, were reduced to approximately half of the activities before perfusion. The activity of the mainly cytosolic enzyme, creatine kinase, was reduced by 58% during 5 h anoxia. In the presence of propranolol, the loss of mitochondrial enzyme activity was significantly reduced, while the loss of creatine kinase activity was unaltered. These findings indicate (1) that loss of respiratory enzyme activity is a consequence of irreversible cellular damage due to anoxia, (2) that a therapeutic level of propranolol conserves respiratory enzyme activity and (3) that the protective action of this level of propranolol is insufficient to conserve creatine kinase activity.     

Inhibition of the Mitochondrial Respiratory Chain by Alanine in Rat Cerebral Cortex

Alanine is a nutritionally nonessential amino acid synthesized by transamination of pyruvate originated from glucose. Alanine is the principal gluconeogenic amino acid because it can originate pyruvate and glucose through the inverse pathway. Considering that it has been suggested that alanine could be used as a dietary supplement in combination with growth hormone in the treatment of undernourished children affected by some inherited metabolic diseases to induce anabolism, the principal objective of the present work was to measure the activities of the mitochondrial respiratory chain complexes and succinate dehydrogenase in brain cortex of Wistar rats subjected to chronic alanine administration from the 6th to the 21st day of life. We also investigated the in vitro effect of alanine on the activities of mitochondrial respiratory chain complexes and succinate dehydrogenase in the same brain structure of 22-day-old rats. The results showed a reduction of Complex I + III and succinate dehydrogenase activities in brain cortex of rats subjected to alanine administration. We also verified that alanine inhibited the in vitro activity of Complexes I + III by competition with NADH. These results indicate that more investigation would be necessary before considering alanine supplementation as a valid adjuvant therapy to sick children with these disorders.     

Geometric specificity of alcohol dehydrogenases and its potential for separation of trans and cis isomers of unsaturated aldehydes.

The geometric specificity of three different alcohol dehydrogenases (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) (from yeast, from horse liver, and from Leuconostoc mesenteroides) in the reduction of trans- and cis-cinnamaldehydes has been investigated. All three enzymes display a remarkable trans specificity: they react with the trans isomer 7 to 647 times faster than with its cis counterpart. Experiments with the enzymatic reduction of 3-phenylpropionaldehyde, a saturated analog of cinnamaldehyde, have revealed that whereas trans-cinnamaldehyde possesses the "right" configuration for the active centers of the alcohol dehydrogenases, the cis isomer apparently does not fit the active centers well. All three alcohol dehydrogenases studied also exhibit a marked trans specificity in the reaction with alpha-methylcinnamaldehyde. The geometric specificity of alcohol dehydrogenases can be used for the production of otherwise hard to synthesize cis isomers of unsaturated aldehydes from their readily available trans counterparts: trans-cinnamaldehyde was irradiated with ultraviolet light (which converted it to a mixture of trans and cis isomers) then treated with NADH and yeast alcohol dehydrogenase (which selectively reduces only trans aldehyde into the alcohol), and finally the mixture of cis-cinnamaldehyde and trans-cinnamyl alcohol was separated easily by preparative column chromatography.     

Pea formaldehyde-active class III alcohol dehydrogenase: common derivation of the plant and animal forms but not of the corresponding ethanol-active forms (classes I and P).

A plant class III alcohol dehydrogenase (or glutathione-dependent formaldehyde dehydrogenase) has been characterized. The enzyme is a typical class III member with enzymatic parameters and substrate specificity closely related to those of already established animal forms. Km values with the pea enzyme are 6.5 microM for NAD+, 2 microM for S-hydroxymethylglutathione, and 840 microM for octanol versus 9, 4, and 1200 microM, respectively, with the human enzyme. Structurally, the pea/human class III enzymes are closely related, exhibiting a residue identity of 69% and with only 3 of 23 residues differing among those often considered in substrate and coenzyme binding. In contrast, the corresponding ethanol-active enzymes, the long-known human liver and pea alcohol dehydrogenases, differ more (47% residue identities) and are also in functionally important active site segments, with 12 of the 23 positions exchanged, including no less than 7 at the usually much conserved coenzyme-binding segment. These differences affect functionally important residues that are often class-distinguishing, such as those at positions 48, 51, and 115, where the plant ethanol-active forms resemble class III (Thr, Tyr, and Arg, respectively) rather than the animal ethanol-active class I forms (typically Ser, His, and Asp, respectively). Calculations of phylogenetic trees support the conclusions from functional residues in subgrouping plant ethanol-active dehydrogenases and the animal ethanol-active enzymes (class I) as separate descendants from the class III line. It appears that the classical plant alcohol dehydrogenases (now called class P) have a duplicatory origin separate from that of the animal class I enzymes and therefore a paralogous relationship with functional convergence of their alcohol substrate specificity. Combined, the results establish the conserved nature of class III also in plants, and contribute to the molecular and functional understanding of alcohol dehydrogenases by defining two branches of plant enzymes into the system.     

Intracellular Hydrogen Transport Systems in Acute Leukaemia

S ummary . A cytochemical study of intra- and extra-mitochondrial dehydrogenase enzymes in human leukaemic lymphoblasts has shown an increase in activity, relative to the small lymphocyte, of those enzymes which are extramitochondrial in location. In contrast, there was no significant increase in the activity of intramitochondrial respiratory dehydrogenases or of an α-glycerolphosphate dehydrogenase which is thought to regulate hydrogen transport across the mitochondrial membrane. The findings are in keeping with a hypothesis that leukaemic blast cells show diminished hydrogen transport to the intramitochondrial respiratory pathway. A compensatory increase in lactate dehydrogenase activity in these cells may signify a degree of metabolic dependence on this enzyme, and its selective inhibition by oxamate analogues may therefore result in a cytotoxic effect.     

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

By crossed immunoelectrophoresis with membrane antiserum, 17 antigens have been detected in fractions from plasma membranes of M. lysodeikticus solubilized with Triton X-100. Absorption tests with protoplasts have demonstrated that eight of the antigens are expressed on the surface. Of these antigens the major one has been identified as a succinylated mannan. Five of the principal immunoprecipitates unaffected by absorption with protoplasts were shown by zymograms to possess the following enzymic activites: succinate dehydrogenase (EC 1.3.99.1), ATPase (EC 3.6.1.3), NADH dehyrogenase (EC 1.6.99.3)(two separate components), and malate dehydrogenase (EC 1.1.1.37). These enzymes or enzyme-complexes are, therefore, not expressed on the outer surface of the protoplast membrane.     

Glucocorticosteroid inhibition of nonphagocytic discharge of lysosomal enzymes from human neutrophils

Glucocorticosteroids and mineralocorticosteroids were tested for their capacity to inhibit the nonphagocytic discharge of two lysosomal enzymes—a cartilage matrix-degrading neutral protease and β-glucuronidase—from highly purified human neutrophils. Lysosomal enzyme discharge from neutrophils adherent to nonphagocytizable, immobilized, heat-aggregated IgG was inhibited by the four glucocorticosteroids—methylprednisolone sodium succinate, triamcinolone acetonide hemisuccinate, para-methasone acetate, and hydrocortisone sodium succinate. These glucocorticoids also inhibited zymosan-induced release of β-glucuronidase from neutrophils that had been pretreated with cytochalasin B in order to completely prevent the onset of phagocytosis. Inhibition by the glucocorticoids of lysosomal enzyme discharge provoked by a soluble divalent cation ionophore was also observed. Neither desoxycorticosterone acetate nor aldosterone hemisuccinate, two mineralocorticosteroids, inhibited lysosomal enzyme release. Similarly, the salt moieties of some of the steroids tested, such as sodium succinate and sodium acetate, failed to elicit an effect on enzyme release. Therefore interference with lysosomal enzyme discharge was restricted to the glucocorticosteroid ring structure. Because interference either with the adherence of neutrophils to immune reactants or with the activities of the discharged lysosomal enzymes by the glucocorticoids could be interpreted as inhibition of lysosomal enzyme release, steroidal effects on these parameters were examined. None of the glucocorticoids tested elicited any significant effects on neutrophil adherence or lysosomal enzyme activity. Thus it appears that glucocorticosteroids are capable of inhibiting directly the nonphagocytic discharge of lysosomal enzymes from human neutrophils.     

Regulation of the activities of the enzymes involved in the metabolism of steroid hormones in rat liver: the effect of administration of anterior hypophyseal hormones and gonadotrophin preparations to hypophysectomized rats.

A crude human hypophyseal extract (HE), as well as human growth hormone (GH), ovine prolactin (PRL) and commercial preparations of ACTH, TSH, pregnant mare's serum gonadotrophins (PMS) and chorionic gonadotrophin (CG) were tested for their ability to induce the activities of cytoplasmic 17 beta-hydroxysteroid dehydrogenase and microsomal delta 4-5alpha-hydrogenase and to repress the activities of microsomal 3alpha- and 3beta-hydroxysteroid dehydrogenases in the liver of hypophysectomized rats. The activity of 17beta-hydroxysteroid dehydrogenase was not affected by any of the administered hormones. For the other enzymes, only PRL was effective in causing changes in the activities; the repressive effect on 3alpha-hydroxysteroid dehydrogenase activity was highly significant (P less than 0.001). These results indicate that PRL is involved in the regulation of at least some of the enzyme activities of hepatic steroid hormone metabolism.     

Membrane association of proline dehydrogenase in Escherichia coli is redox dependent.

The PutA protein, product of the Escherichia coli gene putA, has two functions essential for proline utilization and for the regulation of putP and putA expression: as the peripheral membrane flavoprotein, proline dehydrogenase (EC 1.5.99.8), it transfers electrons from proline to the respiratory chain, and, as a repressor, it controls expression of genes putP and putA in response to proline supply. Association of proline dehydrogenase with the membrane was shown to require the simultaneous presence of the soluble enzyme, membranes, and proline. The kinetics of that association, monitored by following proline oxidation in a coupled enzyme assay system, were not altered when the transmembrane proton gradient generated during proline oxidation was dissipated by a proton ionophore. However, D-lactate or NADH could replace proline as a promoter of proline dehydrogenase-membrane association under anaerobic reaction conditions. These data imply that reduction of proline dehydrogenase and/or a membrane constituent promotes enzyme-membrane association. A biochemical mechanism is suggested whereby the concentration of proline dehydrogenase associated with the respiratory chain would be determined by proline supply.