E2 Isozyme Research Articles

Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm). In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.

Human Aldehyde Dehydrogenase Catalytic Activity and Structural Interactions with Coenzyme Analogs

Km and Vmax values for 10 coenzyme analogs never previously studied with any aldehyde dehydrogenase and NADP+ were compared with those for NAD+ for three human aldehyde dehydrogenases (EC 1.2.1.3); the cytoplasmic E1 (the product of the aldhl gene), the mitochondrial E2 (the product of the aldh2 gene) and the cytoplasmic E3 (the product of the aldh9 gene) isozymes. Structural information on changes in coenzyme-protein interactions were obtained via molecular dynamics (MD) studies with the E2 isozyme and quantum mechanical (QM) calculations were used to study changes in charge distribution of the pyridine ring and relative free energies of solvation of the purine ring in the analogs. E1 showed the broadest substrate specificity and was the only isozyme subject to substrate inhibition, both of which are suggested to be due to the two coenzyme conformations observed previously in the sheep crystal structure. NADP+ selectivity is indicated to be influenced by Glu195 in E1 and E2. Substitutions in the purine ring affected Km but not Vmax, with the changes in Km being dominated by the hydrophobicity of the purine ring as indicted by the QM calculations. Substitutions in the pyridine ring sometimes rendered the coenzymes inactive, with no consistent pattern observed for the three coenzymes. Structural analysis of the coenzyme analog-E2 MD simulations revealed different structural perturbations of the surrounding active site, though interactions with Asn169 and Glu399 were preserved in all cases.

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.

Metabolism of Retinaldehyde and Other Aldehydes in Soluble Extracts of Human Liver and Kidney

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD(+)-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD(+)-dependent retinaldehyde activity was associated with the E1 isozyme (coded for by aldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD(+)-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD(+)-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1 gene) and broad substrate specificity E1 (a member of EC 1. 2.1.3 aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.

Binding and incorporation of 4-trans-(N,N-dimethylamino) cinnamaldehyde by aldehyde dehydrogenase.

4-trans-(N,N-Dimethylamino)cinnamaldehyde (DACA) is a chromophoric substrate of aldehyde dehydrogenase (EC 1.2.1.3) whose fate can be followed during catalysis. During this investigation we found that DACA also fluoresces and that this fluorescence is enhanced and blue-shifted upon binding to aldehyde dehydrogenase. Binding of DACA to aldehyde dehydrogenase also occurs in the absence of coenzyme. Benzaldehyde (a substrate), acetophenone (a substrate-competitive inhibitor), and the substrate-competitive affinity reagent bromoacetophenone interfere with DACA binding. Thus, DACA binds to the active site and can be employed for titration of active aldehyde dehydrogenase. Both E1 and E2 isozymes, which are homotetramers, bind DACA with dissociation constants of 1-4 microM with a stoichiometry of 2 mol DACA/ mol enzyme. The stoichiometry of enzyme-acyl intermediate was also found to be 2 mol DACA/ mol enzyme for both E1 and E2 isozymes. Thus, both enzymes appear to have only two substrate-binding sites which participate in catalysis. The level of enzyme-acyl intermediate remained constant at different pH values, showing that enhancement of velocity with pH was not due to altered DACA-enzyme levels. When the reaction velocity was increased even further by using 150 microM Mg2+ the intermediate level was decreased, suggesting that both increased pH and Mg2+ promote decomposition of the DACA-enzyme intermediate. Titration with DACA permits study of aldehyde substrate catalysis before central complex interconversion.

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme

Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. The properties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substrate specificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; only two substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme, as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which has broad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This report establishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenates betaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an important function in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organisms as a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrial betaine aldehyde dehydrogenase from any mammalian species.

Esterase Isozymes Associated with Bifenthrin Resistance in the Silverleaf Whitefly (Homoptera: Aleyrodidae)

Esterase isozymes associated with bifenthrin-resistant and susceptible strains of the silverleaf whitefly, Bemisia argentifolii (Bellows & Perring), were investigated using electrophoretic techniques. Samples of adults from the parent susceptible strain, parent resistant strain, F1 crosses, and backcrosses were homogenized in sodium phosphate buffer solutions and analysed using PhastSystem™ (IEF). The results showed that all of the test populations had four common esterase isozymes (E1–E4) in the crude homogenate of whitefly adults based on migration distances. However, significant differences occurred between the susceptible and bifenthrin-resistant strains in the quantity of the E3 isozyme, as measured by the band intensity. This result plus enzyme inhibition tests suggested that E3, a carboxylesterase with pi 5.04, was associated with detoxification of bifenthrin. Changes in quantity of the E3 esterase isozyme, measured with image analysis, was correlated with resistance (higher lethal concentrations) to bifenthrin. The E4 isozyme of all strains tested was inhibited by 1 μM eserine, but sensitivity to eserine was not detected in E2 and E3.

Mechanism of inhibition of aldehyde dehydrogenase by citral, a retinoid antagonist.

Low concentrations of citral (3,7-dimethyl-2,6-octadienal), an inhibitor of retinoic acid biosynthesis, inhibited E1, E2 and E3 isozymes of human aldehyde dehydrogenase (EC1.2.1.3). The inhibition was reversible on dilution and upon long incubation in the presence of NAD+; it occurred with simultaneous formation of NADH and of geranic acid. Thus, citral is an inhibitor and also a substrate. Km values for citral were 4 microM for E1, 1 microM for E2 and 0.1 microM for E3; Vmax values were highest for E1 (73 nmol x min-1 x mg-1), intermediate for E2 (17 nmol x min-1 x mg-1) and lowest (0.07 nmol x min-1 x mg-1) for the E3 isozyme. Citral is a 1 : 2 mixture of isomers: cis isomer neral and trans isomer, geranial; the latter structurally resembles physiologically important retinoids. Both were utilized by all three isozymes; a preference for the trans isomer, geranial, was observed by HPLC and by enzyme kinetics. With the E1 isozyme, both geranial and neral, and with the E2 isozyme, only neral obeyed Michaelis-Menten kinetics. With the E2 isozyme and geranial sigmoidal saturation curves were observed with S0.5 of approximately 50 nM; the n-values of 2-2.5 indicated positive cooperativity. Geranial was a better substrate and a better inhibitor than neral. The low Vmax, which appeared to be controlled by either the slow formation, or decomposition via the hydride transfer, of the thiohemiacetal reaction intermediate, makes citral an excellent inhibitor whose selectivity is enhanced by low Km values. The Vmax for citral with the E1 isozyme was higher than those of the E2 and E3 isozymes which explains its fast recovery following inhibition by citral and suggests that E1 may be the enzyme involved in vivo citral metabolism.

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme

N-tosyl-L-phenylalanine chloromethyl ketone, a serine protease inhibitor, identifies glutamate 398 at the coenzyme-binding site of human aldehyde dehydrogenase. Evidence for a second "naked anion" at the active site.

Human aldehyde dehydrogenase isozymes were inactivated by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of chymotrypsin. The inactivation was a first-order process that followed saturation kinetics. NAD and chloral when used together protected against inactivation. In steady-state kinetics, TPCK produced only slope effects versus varied NAD, both slope and intercept effects versus varied glycolaldehyde were produced, indicating that TPCK reacted with the same enzyme form with which NAD reacted. Ki values from steady-state kinetics and saturation kinetics were comparable. Use of [3H]-labeled TPCK showed that inactivation was associated with the incorporation of two molecules of TPCK per molecule of enzyme. The label incorporation occurred into a single tryptic peptide and also into a single chymotryptic peptide of the E1 isozyme. Purification of labeled peptides, followed by sequencing, demonstrated that E398 of aldehyde dehydrogenase was labeled. Reaction of a haloketone, TPCK, with a carboxyl group of E398 indicates that E398 occurs as a "naked anion" within the molecule. This paper constitutes identification of the second (after E268) "naked anion" at the active site of aldehyde dehydrogenase.

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: Comparison of kinetic properties among the three isozymes

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25 oC, the major and minor components of the E3 isozyme catalyzed the reaction with V max of 1.1 and 0.8 μmol NADH min −1 mg −1 protein, respectively, compared to 0.067 and 0.060 μmol NADH min −1 mg −1 protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K m for methylglyoxal of 8.6 μM, the lowest compared to 46 μM for E1 and 586 and 552 μM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with K i values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 μM for E1 and E2 isozymes, respectively.

Two different amino acid substitutions in the ali-esterase, E3, confer alternative types of organophosphorus insecticide resistance in the sheep blowfly, Lucilia cuprina

Two types of organophosphorus (OP) insecticide resistance are associated with reduced `ali-esterase' (E3 isozyme) activity in Lucilia cuprina. The `diazinon' resistance type shows generally greater resistance for diethyl than dimethyl OPs but no resistance to malathion. The `malathion' resistance type shows generally greater resistance for dimethyl than diethyl OPs, low level diazinon resistance, but exceptionally high malathion resistance (600 × susceptible), the last being attributed to hydrolysis of the carboxylester groups which are peculiar to malathion (malathion carboxylesterase, MCE). E3 variants from diazinon resistant strains have previously been shown to have a Gly 137 → Asp substitution that structural modelling predicts is only about 4.6 Å from the γ oxygen of the catalytic serine residue. Here we show that E3 variants from malathion resistant strains have a Trp 251 → Leu substitution predicted to be about 4.3 Å from that serine. We have expressed alleles of the gene encoding both resistance variants of E3 and an OP susceptible variant in a baculovirus system and compared the kinetics of their products. We find that both resistance substitutions reduce ali-esterase activity and enhance OP hydrolase activity. Furthermore the Gly 137 → Asp substitution enhances OP hydrolase activity for a diethyl OP substrate (chlorfenvinphos) more than does the Trp 251 → Leu substitution, which is consistent with the OP cross-resistance patterns. Trp 251 → Leu also reduces the K m for carboxylester hydrolysis of malathion about 10-fold to 21 μM, which is consistent with increased MCE activity in malathion resistant strains. We then present a model in which the malathion carboxylesterase activity of the E3-Leu 251 enzyme is enhanced in vivo by its OP hydrolase activity. The latter activity enables it to reactivate after phosphorylation by malaoxon, the activated form of malathion, accounting for the exceptionally high level of resistance to malathion. We conclude that the two types of resistance can be explained by kinetic changes caused by the two allelic substitutions in the E3 enzyme.

Pathways of ubiquitin conjugation.

The covalent attachment of the polypeptide ubiquitin to proteins marks them for degradation by the ubiquitin/26S proteasome-dependent degradation pathway. This pathway functions in regulating many fundamental processes required for cell viability. Phylogenetic analysis of ubiquitin sequences reveals greater variability among lower eukaryotes and defines essential residues, many of which are conserved among the three ubiquitin-like proteins known to undergo parallel ligation pathways. The hierarchical design of the ubiquitin conjugation mechanism provides great flexibility for the divergent evolution of new functions mediated by this posttranslational modification. Within this hierarchy, a single ubiquitin-activating enzyme provides charged intermediates to multiple targeting pathways defined by cognate ubiquitin carrier protein (E2)/ligase (E3) pairs. Sequence analysis of E2 isozymes shows that the E2 superfamily is composed of distinct function-specific families. The apparent lack of E2/E3 specificity suggested in the literature results from the presence of multiple isozymes within many E2 families and erroneous family assignments based on incomplete data sets. Other apparent inconsistencies are explained by interfamily sequence relationships among some E2 isoforms.

Induction of Ubiquitin Conjugating Enzyme Activity for Degradation of Topoisomerase IIα during Adenovirus E1A-Induced Apoptosis

Topoisomerase (topo) IIα is degraded via polyubiquitination during adenovirus E1A-induced apoptosis in MA1 cells, a derivative of the human epidermoid carcinoma cell line KB. Topo IIα ubiquitination activity in MA1 cells increased nearly 10-fold after induction of E1A in response to dexamethasone. To identify a topo IIα ubiquitination factor(s), the S100 fractions prepared from apoptosis-induced (42 h) and uninduced (0 h) MA1 cells were first fractionated by ubiquitin–Sepharose columns. The ubiquitination activity induced by E1A was predominantly eluted with 20 mM AMP. Further fractionation of the AMP eluates on Resource-Q columns and the thiolester formation of the proteins resolved by electrophoresis with biotinylated ubiquitin revealed that a species of E2 isozyme recovered in the QFT2 fraction increased markedly in MA1 cells after E1A expression. These results indicate that a ubiquitination factor(s) specific to topo IIα is induced during E1A-induced apoptosis in MA1 cells.

Tissue Distribution of Human Aldehyde Dehydrogenase E3 (ALDH9): Comparison of Enzyme Activity with E3 Protein and mRNA Distribution

The tissue distribution of the E3 isozyme of human aldehyde dehydrogenase has been investigated by three methods: enzyme activity assay employing betaine aldehyde as substrate, Western blotting employing E3 isozyme-specific antibodies, and Northern blotting using a human liver E3 cDNA as probe. All three methods showed that E3 isozyme was universally distributed among all tissues tested. The highest levels of the E3 isozyme activity were found in liver, adrenal gland, and kidney. These same tissues also showed highest levels of the E3 protein via the Western blot. This distribution is consistent with the possible physiological role of E3 isozyme in the synthesis of the osmolyte, betaine, and the neurotransmitter, GABA. Northern blot analysis, however, differed from that of enzyme assay and the Western blot in that it showed highest mRNA levels in skeletal and heart muscles, which had low enzyme activities and E3 protein levels.

Cimetidine and other H2-receptor antagonists as inhibitors of human E3 aldehyde dehydrogenase.

The histamine H2-receptor antagonists have been identified as inhibitors of human liver aldehyde dehydrogenase (EC 1.2.1.3) isozymes, E1, E2, and E3. Inhibition was strongest with the E3 isozyme, whose substrates include gamma-aminobutyraldehyde, the aldehyde metabolites of polyamines, and betaine aldehyde. Burimamide, metiamide, cimetidine guanidine, cimetidine, and tiotidine were competitive with aldehyde substrates and noncompetitive with the coenzyme, binding to both the free E3 isozyme and the enzyme-coenzyme binary complex. Cimetidine and tiotidine were the best inhibitors, with Ki values of 1.1 +/- 0.2 microM and 1.0 +/- 0.0 microM, respectively; both are the first ever described potent and selective inhibitors of the E3 isozyme. Examination of the H2-receptor antagonist structures for insight into the moieties accounting for E3 isozyme inhibition pointed to the side-chain polar groups as strongly influencing inhibition, with the cyanoguanidine side chain of cimetidine and tiotidine having the strongest influence. The Ki value of the E3 isozyme for cimetidine was the same as the in vitro dissociation constant for the H2-receptor.

Novel Multiubiquitin Chain Linkages Catalyzed by the Conjugating Enzymes E2EPF and RAD6 Are Recognized by 26 S Proteasome Subunit 5

Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains. Point mutants of ubiquitin (Lys-->Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2EPF catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6. Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 of partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures. In addition, competition studies provided an estimate of Kd < or = 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2EPF retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase.

Human Aldehyde Dehydrogenase E3 Isozyme Is a Betaine Aldehyde Dehydrogenase

The E3 isozyme of human aldehyde dehydrogenase (EC 1.2.1.3), with broad substrate specificity, which also catalyzes dehydrogenation of 4-aminobutyraldehyde, was purified and sequenced recently (1,3). It has been shown during this investigation to have betaine aldehyde dehydrogenase activity. Betaine aldehyde and 4-aminobutyraldehyde activities copurified on six chromatographic columns. Molecular properties of the homogeneous product were identical with those of E3 isozyme. Activity with betaine aldehyde was considerably higher than that with 4-aminobutyraldehyde, the best known substrate. Thus, human E3 isozyme and betaine aldehyde dehydrogenase (EC 1.2.1.8) are the same enzyme.

New human aldehyde dehydrogenases.

Electrophoretic separation techniques of alcohol and aldehyde dehydrogenases have been generally employed for identification of the individual isozyme components. The electrophoretic patterns of alcohol dehydrogenase, a largely cathodal enzyme, are relatively easy to interpret because only a few other proteins migrate towards cathode and different isozymes have different isoelectric points. Aldehyde dehydrogenase, however, is largely an anodal enzyme, which migrates in the direction of the majority of other proteins, large numbers of which superimpose with aldehyde dehydrogenase. Some of these proteins produce gel bleaching during nitroblue tetrazolium / phenazine methosulfate color development and thereby mask some weaker aldehyde dehydrogenase bands. This is the reason why only the major bands of aldehyde dehydrogenase are usually visualized. In addition, there are several different aldehyde dehydrogenases that have the same pI as the major bands and therefore superimpose on isoelectric focusing gels. The superimposals that are now known to occur as a result of work of a large number of investigators (Greenfield and Pietruszko, 1977; Impraim et al., 1982; Jones and Teng, 1983; Palmer and Jenkins, 1985; Santisteban et al., 1985; Forte McRobbie and Pietruszko, 1986; Ryzlak and Pietruszko, 1988 a, b; Kurys et al., 1989; Wang et al., 1990) are shown in Table 1. The best known superimposals occur at pI 5.3 where the human E1 isozyme, human E3 isozyme and the Oriental E2 isozyme are found.

Purification and characterization of a ubiquitin carrier protein kinase from HeLa cells.

Protein ubiquitination plays an important role in ATP-dependent protein turnover, and it also may regulate other cellular events. Covalent attachment of ubiquitin to other proteins is catalyzed by three different enzymes, E1, E2, and E3. We have previously shown that protein ubiquitination can be regulated by phosphorylation. In the present study, we show that 20-kDa E2, an E2 molecular mass isoform, is phosphorylated by a protein kinase from the cytosolic fraction of HeLa cells. This protein kinase was purified by a procedure involving ammonium sulfate precipitation and three column chromatographies (phenyl-Sepharose, Superose gel filtration, and DEAE-Sephacel). Gel-filtration chromatography indicated that the molecular mass of this protein kinase was about 300 kDa. However, SDS/PAGE showed that the purified protein kinase consists of three subunits with molecular masses of 120, 105, and 70 kDa, respectively. The stoichiometry of the phosphorylated 20-kDa E2 isozyme was found to be 0.45 mol of phosphate per mol of protein. The phosphorylation of 20-kDa E2 occurred only at the serine residue. The activity of this protein kinase required the presence of Mg2+; however, the enzyme was inhibited by a high concentration of Mg2+.