Cerebral-cortex hexokinases. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis

1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP(2-). Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP(2-) was the substrate varied. The K(i) for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and betagamma-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP(2-) was varied. AMP inhibition was competitive when MgATP(2-) was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.     

Predominance of the Cytoplasmic Form of Brain Hexokinase in Cultured Astrocytes

Abstract: Astrocytes have been cultured from neonatal rat brain according to the flask culture procedure of Booher and Sensenbrenner. Approximately 80% of the hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1) activity is found in the soluble fraction in homogenates of these cells, in contrast to only 20% of the total activity in the soluble fraction of whole brain homogenates. The hexokinase from the cultured astrocytes has been compared with the cytoplasmic and glucose-6-P-solubilized mitochondrial enzymes from whole brain. In kinetic properties and pH-activity relationships, the glial hexokinase was similar to the cytoplasmic enzyme but different from the mitochondrial enzyme of whole brain. Using immunohistochemical methods for detecting hexokinase localization at the electron microscopic level, most of the cells showed prominent staining of cytoplasmic areas. If the cultured astrocytes are accepted as valid models for astrocytes in situ , these results support the suggestion of Bigl and co-workers that the predominant form of hexokinase in glial cells is the cytoplasmic enzyme.     

Cerebral-cortex hexokinase. Comparison of properties of solubilized mitochondrial and cytoplasmic activities

1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50–80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, K_m(ATP) 0.60mm, K_m(glucose) 0.042mm; peak II, K_m(ATP) 0.66mm, K_m(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, K_m(ATP) 0.56mm, K_m(glucose) 0.048mm; peak II, K_m(ATP) 0.68mm, K_m(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.     

Purification and properties of cytoplasmic and mitochondrial hexokinase from the rabbit brain

The soluble Hexokinase from rabbit brain was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The properties of the purified cytoplasmic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in M.W., pI and electrophoretic mobility. However, the temperature dependence of activity and specificity for several hexose substrates were markedly different.     

Sugar phosphorylation modulates ADP inhibition of maize mitochondrial hexokinase

The preference of maize ( Zea mays L.) mitochondrial hexokinase (EC 2.7.1.1.) for glucose and fructose and the ADP regulation were evaluated. The Michaelis-Menten constants (K_m) varied between 0.02 and 0.09 m M for glucose and from 2 to 6 m M for fructose as substrates. The value of V_max was five times higher in the presence of glucose as compared with fructose in membrane-bound enzyme preparations. It was shown that ADP produced from the reaction inhibits the hexokinase activity (K_i=20–50 μ M ). However, the inhibition was very specific for adenine nucleotide. Only a small inhibition was observed when 1 m M of UDP, CDP or GDP was included in the assay medium. Nevertheless, the ADP inhibition was observed only when glucose was phosphorylated. In assay conditions where fructose serves as substrate, the affinity for ADP decreased by 10-fold (K_i varied between 500 and 1  000 μ M ). These kinetics properties were also observed in partially purified soluble enzyme preparations. These data suggest that the type of hexose bound to the catalytic site modulates the ADP control of maize mitochondrial hexokinase.     

Intracellular distribution of hexokinase in rabbit brain

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence that this particulate fraction also contains endoplasmic vesicles and synaptosomes. Solubilization of the bound hexokinase by different combinations of detergents and metabolites has proved the existence of different hexokinase binding sites. Electron microscopic examination of hexokinase location by immuno-gold labelling techniques confirmed, that hexokinase is indeed predominantly bound to mitochondria but that a significant proportion is also bound to non-mitochondrial membranes. Attempts to quantify this distribution were unsuccessful since different figures were obtained using anti-hexokinase IgG affinity purified on immobilized native or denatured hexokinase. Binding studies of the purified rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitochondria there is another binding site on microsomes. The N-terminal sequence of hexokinase has been shown to be important for mitochondria binding and also for microsome binding. These results suggest that the intracellular localization of hexokinase in rabbit brain is not exclusively mitochondrial and that the metabolic role of this enzyme should be reconsidered by including a binding site on the endoplasmic reticulum.     

Involvement of Porin N,N-dicyclohexylcarbodiimide-Reactive Domain in Hexokinase Binding to the Outer Mitochondrial Membrane

The proportion of hexokinase that is bound to the outer mitochondrial membrane is tissue specific and metabolically regulated. This study examined the role of the N,N-dicyclohexylcarbodiimide-binding domain of mitochondrial porin in binding to hexokinase I. Selective proteolytic cleavage of porin protein was performed and peptides were assayed for their, effect on hexokinase I binding to isolated mitochondria. Specificity of DCCD-reactive domain binding to hexokinase I was demonstrated by competition of the peptides for porin binding sites on hexokinase as well as by blockage hexokinase binding by N,N-dicyclohexylcarbodiimide. One of the peptides, designated as 5 kDa (the smallest of the porin peptides, which contains a DCCD-reactive site), totally blocked binding of the enzyme to the mitochondrial membrane, and significantly enhanced the release of the mitochondrially bound enzyme. These experiments demonstrate that there exists a direct and specific interaction between the DCCD-reactive domain of VDAC and hexokinase I. The peptides were further characterized with respect to their effects on certain functional properties of hexokinase I. None had any detectable effect on catalytic properties, including inhibition by glucose 6-phosphate. To evaluate further the outer mitochondrial membranes role in the hexokinase binding, insertion of VDAC was examined using isolated rat mitochondria. Pre-incubation of mitochondria with purified porin strongly increases hexokinase I binding to rat liver mitochondria. Collectively, the results imply that the high hexokinase-binding capability of porin-enriched mitochondria was due to a quantitative difference in binding sites.     

Glucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase

In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K.K., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 17422-17428; Parry, D.M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). In extending these studies, we have isolated a cDNA clone of hexokinase from a lambda gt11 library of the highly glycolytic, c37 mouse hepatoma cell line. This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons. This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes. The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme. At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in the rat liver glucokinase, a cytoplasmic enzyme. The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold. The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2. This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E. coli. Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed.     

Selective permeability of rat liver mitochondria to purified malate dehydrogenase isoenzymes in vitro.

1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.     

Discrimination of antigenic site and thiol-inhibitor-sensitive site of hexokinase isoenzymes.

Rabbit antiserum was prepared against hexokinase isoenzyme type I which was purified from rat brain mitochondria. The antiserum inhibited the activity of the mitochondrial hexokinase type I as well as that of the cytosolic type I enzyme prepared from rat brain, kidney and spleen. It did not, however, inhibit the activity of type II hexokinase from muscle and spleen or that of the type III enzyme from spleen. The results suggest that all hexokinase type I isoenzymes may have a common antigenic site irrespective of their sources, though their responses to a thiol inhibitor are different.     

Relative levels of hexokinase in isolated neuronal, astrocytic, and oligodendroglial fractions from rat brain

The levels of hexokinase, as well as those of the cytoplasmic glycolytic enzyme lactate dehydrogenase and the mitochondrial tricarboxylic acid cycle enzymes fumarase and citrate synthase, have been determined in whole rat brain and in neuronal, astrocytic, and oligodendroglial fractions isolated from rat brain. Compared with either whole brain or with isolated neurons or astrocytes, oligodendroglia are low in hexokinase content. This provides direct confirmation for the conclusion, based on an electron microscopic immunohistochemical method, that oligodendroglia, compared with other neural structures, contain relatively low levels of this key enzyme of glucose metabolism. Based on this confirmation, it is concluded that the electron-microscopic immunohistochemical procedure provides a valid indication of hexokinase content, and thus that other structures shown to stain weakly by the latter technique (e.g., dendritic terminals of cerebellar granule and Purkinje cells) are, indeed, low in hexokinase activity.     

Differences in catalytic properties between cerebral cytoplasmic and mitochondrial hexokinases.

1. Clear kinetic differences between cytoplasmic and mitochondrial forms of type-I cerebral hexokinase were demonstrated from experiments performed under identical conditions on three (cytoplasmic, bound mitochondrial and solubilized mitochondrial) preparations of the enzyme. 2. Whereas the Michaelis constant for glucose (KmGlc) was consistent, that for MgATP2- (KmATP) was lower in the cytoplasmic than in the two mitochondrial preparations. The substrate dissociation constants (KsGlc and KsATP) were both higher in the cytoplasmic than in the mitochondrial preparations. A further difference in the substrate kinetic patterns was that KmATP=KmATP for the cytoplasmic enzyme, in contrast with the mitochondrial enzyme, where KmATP was clearly not equal to KsATP [Bachelard et al. (1971) Biochem. J. 123, 707-715]. 3. Dead-end inhibition produced by N-acetyl-glucosamine and by AMP also exhibited different quantitative kinetic patterns for the two enzyme sources. Both inhibitions gave Ki values similar or equal to those of Ki' for the cytoplasmic activity, whereas Ki was clearly not equal to Ki' for the mitochondrial activity. 4. All of these studies demonstrated the similarity of the two mitochondrial activities (particulate and solubilized), which were both clearly different from the cytoplasmic activity. 5. The analysis gives a practical example of our previous theoretical treatment on the derivation of true inhibition constants. 6. The results are discussed in terms of the function of cerebral hexokinases.     

Glucose catabolism in brain. Intracellular localization of hexokinase

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.     

Modified properties of hexokinase from heart mitochondria prepared using proteolytic enzyme

Summary Isolation of muscle mitochondria is made easier by using proteolytic treatment of the tissue before homogenization. Normally, the proteolytic enzyme is discarded with the supernatant of the first centrifugation. However, our results show that a fraction of enzyme activity remains associated with mitochondria. As shown in experiments described in this paper, mitochondrial hexokinase from tissue treated or not with the proteolytic enzyme exhibits similar properties except that the solubilized enzyme from protease treated tissue is no longer able to rebind to mitochondrial membrane. This modification of the binding ability of the enzyme results from a partial hydrolysis of hexokinase during solubilization experiments by the proteolytic enzyme. Since, as pointed out here, proteolytic enzyme can remain associated with mitochondria, [either adsorbed on mitochondrial membrane or included in the mitochondrial pellet] its use for the isolation of muscle mitochondria should be avoided.     

Purification of the yeast mitochondrial methionyl-tRNA synthetase. Common and distinctive features of the cytoplasmic and mitochondrial isoenzymes

Yeast-mitochondrial methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNA(Met)-Sepharose, Agarose-hexyl-AMP) to yield to a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl-tRNA synthetase (Mr 85,000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65,000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152,000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA(Met) are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNA(Met). Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes. Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.     

An intact hydrophobic N-terminal sequence is critical for binding of rat brain hexokinase to mitochondria

The N-terminal sequence of rat brain hexokinase (ATP: D-hexose-6-phosphotransferase, EC 2.7.1.1) has been determined to be X-NH-Met-Ile-(Ala, Gln)-Ala-Leu-Leu-Ala-Tyr-, where X is a blocking group on the N-terminal methionine, probably an N-acetyl group. Modification of this hydrophobic N-terminal segment by endogenous proteases in crude brain extracts resulted in loss of the ability to bind to mitochondria, but had no effect on catalytic activity, resulting in the appearance of nonbindable enzyme reported by several previous investigators to be present in purified hexokinase preparations. Similar results can be obtained by deliberate limited digestion with chymotrypsin (cleavage points marked by arrows in sequence above). Both bindable and nonbindable enzyme, the latter generated either by endogenous proteases or with chymotrypsin, have an identical C-terminal dipeptide sequence, Ile-Ala. The great susceptibility of the N-terminus to proteolysis plus the marked effect that its proteolytic modification has on binding of hexokinase to anion exchange or hydrophobic (phenyl-Sepharose) matrices suggest that this N-terminal segment is prominently displayed at the enzyme surface. Epitopes recognized by two monoclonal antibodies which block binding of hexokinase to mitochondria (but have no effect on catalytic activity) have been mapped to a 10K fragment cleaved from the N-terminus by limited tryptic digestion. Thus the binding of hexokinase to mitochondria appears to occur via a "binding domain" constituting the N-terminal region of the molecule, with maintenance of an intact hydrophobic sequence at the extreme N-terminus being critical to this interaction. A resulting specific orientation of the molecule on the mitochondrial surface is considered to be a prerequisite for the observed coupling of hexokinase activity and mitochondrial oxidative phosphorylation.     

Purification and properties of the cytoplasmic hexokinase from rabbit brain

About 90% of the total hexokinase activity in rabbit brain was found to be associated with mitochondria while the remaining part was found in the cytosolic fraction. The soluble enzyme was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The molecular weight of the purified hexokinase was found to be approximately 98,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.2 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses. Mg . ATP2-, among the nucleotide substrates, was the most effective phosphate donor. The properties of the purified cytoplasmatic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in molecular weight, isoelectric point, pH dependence of activity, electrophoretic mobility and affinity for glucose and Mg.ATP2-. However, the temperature dependence of activity, and the specificity for several hexose substrates were markedly different.     

Solubilization,purification, and properties of rabbit brain hexokinase

More than 90% of the total hexokinase activity in rabbit brain was found to be associated with the mitochondrial fraction. The participate enzyme was solubilized in a relatively specific way by glucose 6-phosphate and Triton X-100 and purified to apparent homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and affinity chromatography. The solubilized hexokinase activity has been purified 700-fold in 48% yield with a specific activity of 165 units/mg of protein. The molecular weight was found to be approximately 100,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.3 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses with different affinities. Mg · ATP, among the nucleotide substrates, was the most effective as a phosphate donor. The present results indicate considerable similarity between this enzyme and the other mammalian type I hexokinases.     

Betaine aldehyde dehydrogenase from rat liver mitochondrial matrix

An NAD-linked aldehyde dehydrogenase which in addition to aliphatic and aromatic aldehydes, metabolizes aminoaldehydes and betaine aldehyde, has been purified to homogeneity from male Sprague-Dawley rat liver mitochondria. The properties of the rat mitochondrial enzyme are similar to those of a rat liver cytoplasmic betaine aldehyde dehydrognase and the human cytoplasmic E3 isozyme. The primary structure. of four tryptic peptides were also similar; only one difference in primary structure was observed. The close similarity of properties of the cytoplasmic with the mitochondrial form suggest that the cytoplasmic and mitochondrial betaine aldehyde dehydrogenase may be coded for by the same nuclear gene. Investigation of the mitochondrial form by isoelectric focusing resulted in visualization of multiple forms, different from those seen in the cytoplasm suggesting that the enzyme may be processed in the mitochondria.     

Betaine aldehyde dehydrogenase from rat liver mitochondrial matrix

An NAD-linked aldehyde dehydrogenase which in addition to aliphatic and aromatic aldehydes, metabolizes aminoaldehydes and betaine aldehyde, has been purified to homogeneity from male Sprague–Dawley rat liver mitochondria. The properties of the rat mitochondrial enzyme are similar to those of a rat liver cytoplasmic betaine aldehyde dehydrognase and the human cytoplasmic E3 isozyme. The primary structure. of four tryptic peptides were also similar; only one difference in primary structure was observed. The close similarity of properties of the cytoplasmic with the mitochondrial form suggest that the cytoplasmic and mitochondrial betaine aldehyde dehydrogenase may be coded for by the same nuclear gene. Investigation of the mitochondrial form by isoelectric focusing resulted in visualization of multiple forms, different from those seen in the cytoplasm suggesting that the enzyme may be processed in the mitochondria.