Activation of glutamate dehydrogenase by L-leucine

The activation of glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) by L-leucine has been studied. Apparently homogeneous preparations from ox liver and brain were found to respond similarly. Commercially obtained preparations of the enzyme, which had suffered limited proteolysis during the purification procedure, were shown to behave similarly to preparations which had not suffered such proteolysis when the effects of L-leucine on the oxidative deamination reaction were studied using either NAD+ or NADP+ as the coenzyme. There was also no significant difference in the responses when the reductive reaction was determined with NADPH or with 40 microM NADH. At higher concentrations of NADH (160 microM) the unproteolysed preparations were activated by L-leucine to a considerably greater extent than those which had suffered limited proteolysis. These results accord with the greater sensitivity of the former preparations to inhibition by high concentrations of NADH and the relief of such inhibition by L-leucine. This amino acid was also found to relieve the inhibition of the enzyme by GTP, resulting in an apparent increase in the activation observed in the presence of this nucleotide. In contrast, under the conditions used in this work, the apparent degree of activation by L-leucine was found to be decreased in the presence of the activators ATP or ADP. The presence of high concentrations of NADH (200 microM) potentiated the high substrate inhibition by 2-oxoglutarate, and L-leucine significantly reduced this effect. The effects of L-leucine on the activity of glutamate dehydrogenase thus appear to be composed of a direct effect on the activity of the enzyme together with a relief of high substrate inhibition. The effects of GTP and 2-oxoglutarate in potentiating inhibition by NADH can account for their effects in enhancing the apparent activation by L-leucine. The marked differences in the responses of proteolysed and unproteolysed preparations of the enzyme result from the effects of proteolysis in decreasing the sensitivity to high concentrations of NADH.     

Outer membrane phospholipase A from Acinetobacter sp. HO1-N.

A phospholipase A1 activity that hydrolyzed cardiolipin to triacyl- and diacyl-lysocardiolipin was localized in outer membrane preparations derived from Acinetobacter sp. HO1-N. The specific activity of the enzyme derived from hexadecane-grown cells was 2.5 to 3 times higher than that derived from NBYE-grown cells. An apparent Km of 2.22 mM was determined, although inhibition kinetics resulted at the higher cardiolipin substrate concentrations. Optimal reaction conditions established on metal requirements. Enzyme activity was obligately dependent on Triton X-100 (0.5%) and was inhibited by cationic and anionic detergents. Cardiolipin-specific phospholipase D converted triacyl-lysocardiolipin to lysophosphatidylglycerol and phosphatidic acid. The specific activity of this enzyme was approximately 100 times greater than that reported for other membrane preparations derived from microorganisms.     

Changes in Enzyme Regulation during Growth of Maize: I. Progressive Desensitization of Homoserine Dehydrogenase during Seedling Growth

The sensitivity of homoserine dehydrogenase (EC 1.1.1.3) to inhibition by the feed-back modifier, l-threonine, was examined in preparations derived from etiolated shoots, roots, and lightgrown tissues of Zea mays L. var. earliking. A progressive decrease in enzyme sensitivity was observed during seedling growth. Enzyme derived from internode tissue retained a greater sensitivity to the effector than enzyme derived from apical portions of etiolated shoots, whereas enzyme from root tips was characteristically more sensitive than that prepared from mature cells of the root. Enzyme desensitization occurred rapidly during culture of excised shoots and the activities of both homoserine dehydrogenase and aspartokinase (EC 2.7.2.4) declined during shoot culture under a variety of conditions. The initial enzyme levels and the characteristic sensitivity of homoserine dehydrogenase were preserved during culture at 5 to 7 C, but desensitization was not prevented by inclusion of cycloheximide in the culture medium.Results of control experiments provide evidence that desensitization occurs in vivo. No alteration of the enzyme properties was detected during extraction or concentration of sensitive or insensitive enzyme or during coextraction of enzyme from mixed populations of different age shoots; nor was a differential distribution of inhibitors or activators indicated during assay of mixed preparations. The change in enzyme sensitivity was apparent under a variety of assay conditions and was not accompanied by changes in the apparent affinity of the enzyme for the substrate, homoserine. It is suggested that systematic changes in the regulatory characteristics of certain enzymes could be an important level of metabolic regulation during cellular differentiation.Three forms of maize homoserine dehydrogenaase were detected after acrylamide gel electrophoresis of samples derived from 72-hr shoots. Similar analysis of samples from older shoots revealed a broad asymmetric band of enzyme activity, suggesting that changes in the relative distribution of specific forms of the enzyme could be related to the growth-dependent changes in the sensitivity of maize homoserine dehydrogenase.     

The interaction of phospholipid membranes and detergents with glutamate dehydrogenase.

1. The interaction of beef liver glutamate dehydrogenase with cardiolipin from both beef liver mitochondria and beef heart mitochondria, with phosphatidylcholine from both beef liver mitochondria and egg-yolk, and with beef brain phosphatidylserine was investigated by steady-state kinetic methods. 2. the phosphatidylcholine did not inhibit the enzyme under a wide range of conditions. The cardiolipins and phosphatidylserine inhibited the enzyme. The inhibition by these lipids was found to diminish with time if the lipids were prepared and the reaction was studied in either phosphate or Tris buffers, but in zwitterionic buffers these lipid brought about a rapid, reversible inhibition which remained stable with time for at least 150 min. 3. The kinetic type of the inhibition was difficult to determine because of variation between lipid sonicates. Complex mixed types of inhibition were found with cardiolipin, and with phosphatidylserine the inhibition approximated to a non-competitive interaction with Ki(app) values varying between (0.9-6.1) x 10(-6)M. 4. The extent of inhibition decreased with increasing pH and with increasing ionic strength. Basic proteins, such as cytochrome c, show a higher affinity for the anionic membranes and can dissociate the enzyme-lipid complexes. Cosonicates of the cardiolipin and phosphatidylcholine inhibited the enzyme, the extent of inhibition increasing in proportion to the amount of acidic lipid. 5.Sodium dodecylsulphate causes a time-dependent inhibition of the enzyme. The kinetics of this effect and its variation with detergent concentration were studied. 6. The relationship of these observations to the structure and function of the enzyme is discussed. It is suggested that their apparent regulation of the enzyme by oestrogens and other small molecules is due to their binding in vitro at sites on the enzyme designed for binding cardiolipin, when the enzyme is functioning in vivo. The association of the enzyme oligomer in vitro may, for similar reasons, be an artifact.     

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.Special issue dedicated to Dr. Santiago Grisolia.     

Role of L-threonine dehydrogenase in the catabolism of threonine and synthesis of glycine by Escherichia coli.

The enzyme L-threonine dehydrogenase was demonstrated in extracts of Escherichia coli K-12, and was shown to be the first enzyme of the pathway converting threonine to glycine. The enzyme was induced by L-leucine, but not by its substrate, L-threonine. The metabolic significance of leucine as a catabolic signal for amino acid degradation is considered.     

L-leucine auxotrophy in Bifidobacterium globosum

The enzymes involved in the biosynthetic pathway of L-leucine were studied in plasmid-negative and plasmid-positive clones derived from the RU 809 strain of the Bifidobacterium globosum species. The growth of plasmid-positive clones in synthetic medium required L-leucine. We have shown that no detectable activity of the beta-isopropylmalate dehydrogenase enzyme was present in plasmid-positive clones, whereas detectable and significant activity of this enzyme was found in plasmid-negative clones. The lack of activity of the beta-isopropylmalate dehydrogenase enzyme is considered responsible for the L-leucine auxotrophy in the plasmid-positive clones.     

Threonine deaminase from Escherichia coli: feedback-hypersensitive enzyme from a genetic regulatory mutant.

A mutation, ilvA538, in the gene coding for the biosynthetic L-threonine deaminase of Escherichia coli K-12 has previously been demonstrated to have pleiotropic regulatory effects leading to low and invariant expression of some of the isoleucine-valine biosynthetic enzyme, and altered expression of the branched-chain aminoacyl-tRNA synthetases. Strain PS187, which carries the ilvA538 allele, has a partial growth requirement for L-isoleucine and is characterized by a sensitivity to growth inhibition by L-leucine. The experiments reported here demonstrate that the L-threonine deaminase produced by strain PS187 is hypersensitive to inhibition by the pathway end product L-isoleucine. In addition, L-leucine, which acts at relatively high concentrations in vitro as an inhibitor of L-threonine deaminase from the wild type, is a more potent inhibitor of the activity of the mutant enzyme. Forty-six derivatives of strain PS187 were isolated as spontaneous mutants resistant to the growth-inhibitory effects of L-leucine. Two of these, strains MSR14 and MSR16, produce an L-threonine deaminase that is more resistant than the wild type to L-isoleucine inhibition, and intermediate between the wild type and strain PS187 with respect to L-leucine inhibition. Strains MSR14 and MSR16 produce L-threonine deaminase and dihydroxyacid dehydrase, the ilvD gene product, at the low levels characteristic of the parent strain. Other L-leucine-resistant derivatives of strain PS187 produce higher levels of the feedback-hypersensitive L-threonine deaminase. Thus, the sensitivity to growth inhibition by L-leucine observed with strain PS187 appears to be related both to the hypersensitivity of L-threonine deaminase to inhibition of catalytic activity and to the low level of ilv gene expression. The results reported here indicated that L-threonine deaminase is structurally altered in strain PS187, and thus provide further support for the proposal that L-threonine deaminase participates as a genetic regulatory element for the expression of the branched-chain amino acid biosynthetic enzymes.     

Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms: implications for biologic function

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.     

The role of residual phospholipids and copurified proteins in the reconstitution of bovine heart mitochondrial ATPase complex

The reactivation of mitochondrial ATPase by acidic and isoelectric phospholipids was studied comparatively with two purified enzyme preparations exhibiting different gel electrophoretic patterns: the preparation of Serrano et al. (1976, J. Biol. Chem. 251, 2453-2461) and the complex V of Galante et al. (1979, J. Biol. Chem. 254, 12372-12379). Isoelectric phosphatidylcholine liposomes showed marked differences in affinity for the two ATPase complexes and produced different maximal reactivations, whereas no significant differences were found with negatively charged liposomes. Analysis of residual phospholipids associated with the two ATPase preparations revealed a greater relative cardiolipin content in complex V. It is proposed that the different patterns of reactivation of the two ATPase preparations by isoelectric phospholipids result from different contents in residual cardiolipin and adenine nucleotide carrier.     

Preventive effect of clofibrate on carnitine palmitoyltransferase I inhibition and mitochondrial membrane phospholipid depletion induced by galactosamine

We have previously reported that a D-galactosamine injection induces a decrease of carnitine palmitoyltransferase I activity correlated with a depletion of total phospholipid content in the mitochondrial membrane. The impact of a short-term clofibrate treatment on these membrane alterations is investigated, i.e., the kinetic properties of carnitine palmitoyltransferase I, including its sensitivity to malonyl-CoA and mitochondrial membrane content of the various phospholipids. A 4-day clofibrate treatment increases by 42% the apparent Km value of carnitine palmitoyltransferase I for palmitoyl-CoA, while the sensitivity of the enzyme to malonyl-CoA appears slightly decreased. Simultaneously, the cardiolipin content is increased by 70% in the mitochondrial membrane, whereas the phosphatidylethanolamine and phosphatidylcholine contents remain almost unaffected. This 4-day clofibrate treatment prevents the inhibition of carnitine palmitoyltransferase I activity subsequent to galactosamine administration but induces an increase in the apparent Km value for palmitoyl-CoA and a decrease of the sensitivity of the enzyme to malonyl-CoA. The contents of phospholipids which are decreased by galactosamine (phosphatidylcholine, -21%; phosphatidylethanolamine, -29%; cardiolipin, -40%) regain the control values when galactosamine administration is preceded by a clofibrate treatment. The data suggest that the clofibrate treatment counteracts the inhibition of activity of carnitine palmitoyltransferase I through the maintenance of mitochondrial membrane integrity.     

Rat striatal synaptosomes as a model system for studying the inhibition of dihydropteridine reductase activity

The distribution of dihydropteridine reductase between soluble and particulate fractions in synaptosomes parallels that of lactate dehydrogenase, but not monoamine oxidase. Ki and I50 values for inhibitors obtained with the enzyme-rich P2 fraction and its twice-washed fraction (P2W2) were essentially the same, and were similar to those obtained with highly purified human liver enzyme. Dihydropteridine reductase inhibitory potency of multi-ring compounds containing a catechol-moiety was greater than that of single ring catecholic compounds, which in turn was greater than that of p-hydroxy-phenolic compounds. The P2 fraction of rat striatal synaptosomal preparations may serve as a convenient source of dihydropteridine reductase for studying the inhibition of this enzyme.     

Association of spin-labelled cardiolipin with dimyristoylphosphatidylcholine-substituted bovine heart cytochrome c oxidase. A generalized specificity increase rather than highly specific binding sites

The endogeneous lipid of bovine heart cytochrome c oxidase has been replaced by dimyristoylphosphatidylcholine using cholate-mediated exchange. The lipid-substituted preparation contained less than 1 mole cardiolipin per mole enzyme and possessed full oxidative activity. The association of spin-labelled cardiolipin with such lipid-substituted cytochrome oxidase preparations has been assayed using ESR spectroscopy. An average relative association constant 5.4-times that for phosphatidylcholine is obtained for cardiolipin. Measurements on preparations with increasing contents of unlabelled cardiolipin, introduced during lipid exchange, reveal that this selectivity corresponds to a generalized increase in specificity for all lipid association sites on the protein.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values <6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.     

L-Lactate dehydrogenase from leaves of higher plants. Kinetics and regulation of the enzyme from lettuce (Lactuca sativa L).

1. L-Lactate dehydrogenase from lettuce (Lactuca sativa) leaves was purified to electrophoretic homogeneity by affinity chromatography. 2. In addition to its NAD(H)-dependent activity with L-lactate and pyruvate, the enzyme also catalyses the reduction of hydroxypyruvate and glyoxylate. The latter activities are not due to a contamination of the enzyme preparations with hydroxypyruvate reductase. 3. The enzyme shows allosteric properties that are markedly by the pH. 4. ATP is a potent inhibitor of the enzyme. The kinetic data suggest that the inhibition by ATP is competitive with respect to NADH at pH 7.0 and 6.2. The existence of regulatory binding sites for ATP and NADH is discussed. 5. Bivalent metal cations and fructose 6-phosphate relieve the ATP inhibition of the enzyme. 6. A function of leaf L-lactate dehydrogenase is proposed as a component of the systems regulating the cellular pH and/or controlling the concentration of reducing equivalents in the cytoplasm of leaf cells.     

Inhibition of 3-phosphoglycerate dehydrogenase by l-serine

1. l-Serine was shown to be a highly specific inhibitor of 3-phosphoglycerate dehydrogenase. 2. 3-Phosphoglycerate dehydrogenase is cold-labile with respect to its catalytic activity and to sensitivity to serine. 3. l-Serine protects the catalytic site as well as the inhibitor site. 4. Glycerol protects the catalytic site as well as the inhibitor site. 5. Serine acts as a ;classical' non-competitive inhibitor of fresh preparations of 3-phosphoglycerate dehydrogenase. 6. ;Aged' preparations when assayed at pH6.5 show sigmoid inhibition curves at saturating substrate concentrations. 7. A generalized model is advanced to account for the variation of the catalytic activity and the inhibitory effect of l-serine with time and conditions. 8. The possibility that the sigmoid kinetics of inhibition observed are an artifact of isolation is discussed.     

Purification of xanthine dehydrogenase from rat liver: a rapid procedure with high enzyme yields

Xanthine dehydrogenase (EC 1.2.1.37) was purified approximately 1000-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (greater than 20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis.     

Protein kinase activity at the inner membrane of mammalian mitochondria.

This paper reports on the discovery of a protein kinase activity associated with the inner membrane of mammalian mitochondria. The enzyme does not respond to addition of cyclic AMP or cyclic GMP and has a preference for whole histone as phosphate acceptor. Some standard assay systems for the cyclic nucleotide-dependent cytosol protein kinases would be unable to pick up this activity of the orthophosphate concentration is higher than 25 mM and the pH or the assay lower than pH 6.5. The enzyme described here has an apparent pH optimum of 8.5. Activity in liver mitochondria is not evident unless the mitochondria are disrupted by either sonication or freezing and thawing. Distribution of kinase activity in centrifugal fractions of both liver and heart mitochondrial sonicates was parallel to that of the two inner membrane marker enzymes succinic dehydrogenase and cytochrome oxidase and quite different from that of the matrix enzyme malic dehydrogenase. Experiments with preparations enriched in outer or inner membranes confirmed the contention that this enzyme is located on the inner membrane. Since disruption of the inner membrane by a freeze-thaw treatment (after the outer membrane had been disrupted by swelling in phosphate) was necessary for full expression of activity by this enzyme, the tentative conclusion was reached that substrate is accepted only from the matrix side of the inner membrane.