Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.     

Molecular cloning of starch synthase I from maize (W64) endosperm and expression in Escherichia coli

Vertebrate myelin is enriched in the lipid galactocerebroside (GalC) and its sulfated derivated sulfatide. To understand the in vivo function of these lipids, we analyzed myelination in mice that contain a null mutation in the gene encoding UDP-galactose:ceramide galactosyltransferase, the enzyme responsible for catalyzing the final step in GalC synthesis. Galactolipid-deficient myelin is regionally unstable and progressively degenerates. At postnatal day 30, demyelination is restricted to the midbrain and hindbrain, but by postnatal day 90, it spreads throughout the central nervous system. Activated microglial cells and reactive astrocytes appear with the loss of myelin in older animals. Nonetheless, major myelin protein gene mRNA levels are normal throughout the life of these animals, suggesting that widespread oligodendrocyte death is not the primary cause of demyelination. The developmental switch in myelin-associated glycoprotein isoform expression, however, does not occur normally in these mice, suggesting an alteration in oligodendrocyte maturation. Taken together, these findings indicate that GalC and sulfatide are required for the long-term maintenance of myelin and that their absence may have subtle effects on the development of oligodendrocytes.     

Purification and characterization of two forms of urokinase

Affinity chromatography on agmatine-Sepharose was used for the separation of two active forms of urokinase (EC 3.4.99.26) from partially purified human urinary urokinase. The approximate molecular weight of the heavier form was 47 000 and of the lighter 33 400. Both forms were homogeneous by sodium dodecyl sulfate gel electrophoresis and by 3H-labeled diisopropylphosphorofluoridate and 14C-labeled p-nitrophenyl-p'-guanidinobenzoate incorporation studies. The 33 400 mol. wt. form had a single chain, and the 47 000 mol. wt. form had two chains (33 100 and 18 600 mol. wt.) linked by disulfide bonds. The specific activity of the heavier form was 104 000 CTA units/mg protein, compared with 226 000 units/mg for the lighter form but the activities per mmol of active site (molar activities) of the two forms were almost identical (9.6-10(9) and 10.2-10(9) CTA units/mmol). Isoelectric focusing on gels showed that the 47 000 material contained one major subform with a pI of 8.60 and a minor subform with a pI of 8.90, while the 33 400 material had three major subforms with pI values of 8.35, 8.60 and 8.70, respectively, and a minor subform with a pI of 8.05. 3H-labeled diisopropylphosphorofluoridate incorporation studies revealed an active-site serine residue in the heavy chain.     

Purification and characterization of animal porphobilinogen synthases. I. Bovine liver porphobilinogen synthase

Porphobilinogen synthase was purified from ox liver by ammonium sulfate fractionation, heat denaturation and column chromatography (purification: 400-fold; specific activity 4.72 nkat). The molecular weight of the native enzyme obtained by thin-layer gel filtration is about 280 000. Using 8M urea in the presence of dithiothreitol as reducing agent, the molecule breaks down into 8 subunits of molecular weight 36 000 (dodecylsulfate gel electrophoresis); the preparation of aminoethylated subunit is described. According to the above-mentioned molecular weight and to the above-mentioned molecular weight and to the quantitative amino acid analysis after total hydrolysis, the following compositon of the enzymes subunit was calculated ASX23-25 Thr7 Ser23-24 Glx29-31 Pro22-23 Gly22-24 Ala36-37 Val23-26 Met7 Ile9 Leu34-35 Tyr10 Phe11-12 Lys11-12 Cys6-7 His6-8 Arg22 Trp1-2. The subunits, having two free sulfhydryl groups, therefore consists of a chain of about 306 amino acids. The Dansyl-Edman procedure did not enable identification of any free N-terminal amino acid. The acyl group blocking the N-terminus is an acetyl group. It was identified, after hydrazinolysis of the enzyme, by means of chromatographic comparison with 1-formyl-2-dansyl-hydrazine and 1-acetyl-2-dansylhydrazine, whose syntheses and UV spectra are described.     

Purification and characterization of phosphatidylglycerolphosphate synthase from Schizosaccharomyces pombe

The enzyme CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase (phosphatidylglycerolphosphate synthase; PGPS4; EC 2.7.8.5) is located in the mitochondrial inner membrane and catalyzes the committed step in the cardiolipin branch of phospholipid synthesis. Previous studies revealed that PGPS is the most highly regulated enzyme in cardiolipin biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. In this work, we report the purification to homogeneity of PGPS from S. pombe. The enzyme was solubilized from the mitochondrial membrane of S. pombe with Triton X-100. The solubilized enzyme, together with the associated detergent and intrinsic lipids, had a molecular mass of 120 kDa, as determined by gel filtration. The enzyme was further purified using salt-induced phase separation, gel filtration, and ionic exchange, hydroxylapatite, and affinity chromatographies. The procedure yielded a homogeneous protein preparation, evidenced by both SDS-polyacrylamide gel electrophoresis (PAGE) and agarose isoelectric focusing under nondenaturing conditions. The purified enzyme had an apparent molecular mass of 60 kDa as determined by SDS-PAGE. The enzyme showed a strong dependence on lipid cofactors for activity in vitro. While both phosphatidic acid and CDP-diacylglycerol appeared to be activators, the most significant activation was observed with cardiolipin. The possible physiological significance of the lipid cofactor effect is discussed. This is the first purification of a eucaryotic PGPS enzyme to date, and the first purification of a phospholipid biosynthetic enzyme from S. pombe.     

Purification and characterization of bovine heart glycogen synthase kinase-3

Glycogen Synthase Kinase-3 (GSK-3) was isolated from bovine heart tissue extracts by a procedure involving ammonium sulfate fractionation, followed by chromatography on phosphocellulose, Cibacron blue 3GA-agarose, DEAE-Sephacel, CM-Sepharose, heparin-agarose, myelin basic protein-Sepharose, and LiChrospher 1000 C00-. GSK-3 was identified by its activation of protein phosphatase-1i (PP-1i). The purified enzyme had a specific activity of 25,500 units of protein phosphatase-1i activated/mg protein. The enzyme is an asymmetric monomeric protein of 53 kDa. The molecular size and retention of activity after autophosphorylation indicated that the isolated enzyme was the GSK-3 alpha-isoform.     

Biochemical and molecular characterization of a novel starch synthase from potato tubers

An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N-terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule-bound starch synthase II (GBSSII) of pea embryos. When expressed in Escherichia coli, the mature protein has starch synthase activity. The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another.     

Purification and characterization of human topoisomerase I mutants

A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.     

Purification, separation and characterization of phosphoglucomutase and phosphomannomutase from maize leaves

BACKGROUND/AIMS: Reactive oxygen species play an essential role in necro-inflammatory processes. Therefore, the aim of the present studies was to investigate the effect of exogenous and endogenously produced H2O2 on the phagocytic capacity and glucose release of perfused cirrhotic rat livers in comparison with that on the controls. METHODS: Complete septal cirrhosis was achieved by oral treatment of rats with thioacetamide for 6 months. The phagocytic capacity of the perfused livers was measured by the uptake of colloidal carbon. During the continuous perfusion with colloidal carbon, either H2O2 or benzylamine was added to the perfusion medium for a limited time period. The latter functioned as an endogenous H2O2 donor. RESULTS: In control rats exogenous and endogenously produced H2O2 caused a transient stimulation of the hepatic colloidal carbon uptake as well as of the glucose release. Inhibition of the catalase by aminotriazol doubled the changes evoked by H2O2, whereas blockade of the Kupffer cells by GdCl3 drastically reduced its stimulatory effect. Cirrhotic livers took up less colloidal carbon and released lower amounts of glucose than the controls when stimulated by exogenous H2O2. The inhibition of the nitric oxide synthetase augmented the H2O2-induced effect in controls as well as in the cirrhotic livers by 250% and 620% (colloidal carbon uptake) and 340% and 760% (glucose release), respectively. The blockade of the eicosanoid production by indomethacin and caffeic acid drastically increased the glucose release and the colloidal carbon uptake in controls and, in absolute terms, to a lesser extent in cirrhotic livers. Endogenous H2O2 produced by the addition of benzylamine stimulated the colloidal carbon uptake and glucose release in livers from both groups. The inhibition of the lipoxygenase increased both parameters, whereas different effects were elicited by the addition of superoxide dismutase in controls and cirrhotic livers. CONCLUSION: The maximum uptake of colloidal carbon and glucose release, measured after stimulation by H2O2, was lower in cirrhotic livers than in controls, thus indicating a lowered phagocytic capacity of Kupffer cells and altered glycogenolytic response of the hepatocytes in cirrhotic livers. The use of various effectors provided evidence that superoxide anions, nitric oxide and, possibly, arachidonic acid are involved in the signal transduction between Kupffer cells and hepatocytes when stimulated by exogenous or endogenously produced H2O2. This signalling mechanism seems to be impaired in cirrhotic livers.     

Purification and characterization of a nitric-oxide synthase from rat liver mitochondria

The biosynthesis of nitric oxide (NO.) in different cell types occurs concomitantly with the conversion of L-arginine to L-citrulline by the enzyme nitric-oxide synthase (NOS). NO. has been identified as a major participant in a number of basic physiological functions such as neurotransmission, vasodilation, and immune response. At the subcellular level, mitochondria have been identified as targets for NO.; however, to date, no unambiguous evidence has been presented to identify these organelles as sources of NO.. In this study, a NOS was isolated to homogeneity from Percoll-purified rat liver mitochondria. Kinetic parameters, molecular weight, requirement of cofactors, and cross-reactivity to monoclonal antibodies against macrophage NOS suggest similarities to the inducible form. However, the constitutive expression of the mitochondrial enzyme and its main membrane localization indicate the presence of either a distinctive isoform or a macrophage isoform containing posttranslational modifications that lead to different subcellular compartments. The detection of NADPH-oxidizing activities and a production of superoxide anion catalyzed by mtNOS and recombinant cytochrome P450 reductase were consistent with the sequence homology reported for these two proteins. Given the role of NO. as cellular transmitter, messenger, or regulator, the presence of a functionally active mitochondrial NOS may have important implications for the intermediary metabolism.     

Human pancreatic alpha-amylase. I. Purification and characterization

alpha-Amylase was extracted from human pancreas and purified by using ammonium sulfate fractionation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography. The enzyme was shown to be homogenous by three different criteria: polyacrylamide disc gel electrophoresis, SDS polyacrylamide gel electrophoresis and analytical ultracentrifugation. The values of SO20,w, D20,w, v, and frictional ration of the enzyme were calculated to be 5.01S, 7.56D, 0.718 ml g-1 and 1.10, respectively. The molecular weight of the alpha-amylase was determined by three different methods: sedimentation velocity-diffusion, conventional sedimentation equilibrium and SDS polyacrylamide gel electrophoresis and was found to be 57,850; 50,100 and 53,200 g mole-1, respectively (average value 53,700). The amino acid composition of the enzyme was determined and compared with those of alpha-amylases from various other sources.     

Purification and characterization of a novel cysteine synthase isozyme from spinach hydrated seeds

A novel type of cysteine synthase (CSase, EC 4.2.99.8) isozyme, designated as CSase 1', was purified to homogeneity from hydrated spinach seeds. The enzyme had a molecular weight of 68,000 and consisted of two identical subunits of M(r), 34,000. The apparent K(m) for O-acetyl-L-serine was 8.33 mM and that for sulfide was 0.66 mM. The activity of CSase 1' was maintained when it was treated at 60 degrees C for 1 min. This novel enzyme was similar to CSases 1, 2, and 3 already purified from spinach leaves, in results of double immunodiffusion, molecular weight, subunit composition, K(m) values for O-acetyl-L-serine and sulfide, and heat stability. On the other hand, N-terminal amino acid sequence, effects of immunotitration, pH optimum, and effects of hydroxylamine on purified CSase 1' were different from those of the other CSases. Furthermore, it was found that CSases 2S and 3S isolated from hydrated spinach seeds were identical with the CSases 2 and 3 reported previously. It was also disclosed that CSases 1, 2, and 3 were localized in chloroplasts, cytosol, and mitochondria, respectively.     

Purification of the stress protein alpha B-crystallin and its differentially phosphorylated forms

The subsolubilizing alterations caused by a series of alkyl glucosides (alkyl chain lengths ranging from C8 to C12) in unilamellar phosphatidylcholine (PC) liposomes were investigated. The surfactant to phospholipid molar ratios (RE) and the normalized bilayer/aqueous phase partition coefficients (K) were determined by monitoring the increase of the fluorescence intensity of liposome suspensions due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles to the bulk aqueous phase. Given that the free surfactant concentrations was always lower than the critical micelle concentration (CMC) of the surfactant tested we may assume that the surfactant-liposome interactions were mainly ruled by the action of surfactant monomers. In general terms, the decrease in the surfactant alkyl chain length (or the rise in the surfactant CMC) resulted in an increase in the ability of these surfactants to alter the permeability of liposomes and, inversely, in an abrupt decrease in their affinity with these bilayers structures. The overall balance of these opposite tendencies shows that at the two interaction levels studied (50 and 100% of CF release) the nonyl and the octyl glucoside showed, respectively, the highest ability to alter the release of the CF trapped in bilayers (lowest RE values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values).     

Purification and characterization of two forms of microsomal cytochrome b5 from sheep lung

Two forms of cytochrome b5 were purified from detergent solubilized sheep lung microsomes by three successive DEAE-cellulose, Sephadex G-100 and Sephadex G-200 column chromatographies. The specific contents of cytochromes b5-I and b5-II were determined to be 45.4 and 43.8 nmol b5/mg protein, which represented up to 567 and 547-fold purification compared with that of the lung microsomes. The most striking difference between b5-I and b5-II was observed in their elution pattern from the third DEAE-cellulose column. Cytochromes gave one protein band with a Mr of 16400 +/- 500 on SDS-PAGE. Both forms of reduced b5 showed a major peak at 423 nm while reduced b5-I had two minor peaks at 527 and 556 and reduced b5-II gave two well-defined peaks at 526 and 555 nm. The ability of the purified b5-I and b5-II fractions to transfer the electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. Apparent Km, 0.055 microM, of b5-II was found to be 38% lower than that of b5-I. In addition, cytochrome b5-I was found to be more sensitive to heat treatment than b5-II when cytochromes were subjected to 62 degrees C for varying periods of time and the coupling of b5 reduction to cytochrome c reduction was determined. These results may indicate that two forms may exist in lung endoplasmic reticulum.     

Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity

A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389). In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide. An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75. The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity. Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel. The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold. These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5. The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity. Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol. Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 microM bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A2, had no significant effect on the enzyme activity. The enzyme activity was completely abolished in the presence of greater than 773 microM Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 microg/ml heparin. In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A2.     

Purification and characterization of NADH-dependent glutamate synthase from the silkworm fat body (Bombyx mori)

NADH-dependent glutamate synthase (Nadh-Gogat; EC 1.41.14) was purified 766-fold from the fat body of 5th instar larvae of the silkworm with a final specific activity of 13.8 units/mg protein by a produce including ammonium sulfate fraction, Q-Sepharose HP ion exchange column chromatography, Blue Sepharose FF affinity column chromatography and Superdex 200 HR gel filtration. The purified enzyme yielded a single band corresponding to a molecular mass of 195kDa by SDS-polyacrylamide gel electrophoresis. Molecular mass of the native enzyme was estimated to be 190 kDa by Superdex 200HR gel filtration, suggesting that the enzyme is composed of a monomeric polypeptide. The enzyme showed an absorption spectrum with maximum values at 272, 375, and 435 nm, suggesting the presence of a flavin prosthetic group in the enzyme. The N-terminal amino acid sequence showed a high similarity to those of other GOGATs from plants, yeast and bacteria, but no similarity to other known proteins was detected. The enzyme exhibited a strong specificity to the electron donor and substrates; NADH as electron donor, 2-oxoglutarate as amino acceptor and glutamine as amino donor were essential for the catalytic activity. The optimum pH was around 7.5, at which Km values for 2-oxoglutarate, glutamine and NADH were 17, 220 and 5.7 micro M, respectively. Azaserine, 6-diazo-5-oxonorleucine and p-chloromercuribenzoic acid were strong inhibitors of the activity. These result show that NADH-GOGAT in the silkworm fat body strongly resembles other eukaryotic NADH-GOGATs, suggesting that it plays a significant role in ammonia assimilation in the same manner as the other enzymes.     

Cloning of a Streptococcus sobrinus oligo-isomaltosaccharide synthase gene and characterization of its product

A Streptococcus sobrinus gene coding for a glucosyltransferase (GTF)-S was cloned into Escherichia coli, using the bacteriophage lambda L47.1 and the plasmid vector pACYC184. The MD124 clone obtained expressed a 155 kDa GTF-S which did not react with any antisera against GTF-S1, -S2 and -I enzymes. The recombinant enzyme (designated rGTF-S3) was homogeneously purified from the MD124 cell-extract and characterized. The purified rGTF-S3 synthesized primer-independently alpha-1,6-linked linear oligosaccharides from sucrose. The dependence upon the sucrose concentration was diphasic, and the respective Km values were 1.3 and 25 mM. The properties except the Km values were similar to those of oligo-isomaltosaccharide synthase from S. sobrinus AHT.     

Purification and characterization of laccase from Chaetomium thermophilium and its role in humification

Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process. C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45 degreesC both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70 degreesC and had half-lives of 24 and 12 h at 40 and 50 degreesC, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.     

Purification and characterization of a high molecular weight histone deacetylase complex (HD2) of maize embryos

The dynamic state of core histone acetylation is maintained by histone acetyltransferases and deacetylases. In germinating maize embryos, four nuclear histone deacetylases can be distinguished. From a chromatin fraction prepared at 72 h after start of embryo germination, we have purified the nuclear histone deacetylase HD2 to homogeneity. Using a sequence of chromatographic steps, we achieved the purification of an enzymatically active high molecular weight protein complex with an apparent molecular mass of 400 kDa, as determined by gel filtration chromatography. The purified enzyme was characterized in terms of enzymatic and kinetic properties, and sensitivity to several histone deacetylase inhibitors. In SDS-polyacrylamide gels, HD2 split into three polypeptides of 45, 42, and 39 kDa, suggesting that the native enzyme is a multimer-protein complex. Electrophoresis under nondenaturing conditions in combination with second dimension SDS-gel electrophoresis indicated that all three protein components of the HD2 complex were enzymatically active. Polyclonal antibodies against each of the three polypeptides were raised in rabbits. Each antiserum reacted with all three polypeptides on Western blots, suggesting that p45, p42, and p39 are highly homologous. This homology was confirmed by amino acid sequencing of peptides generated from each of the three HD2 components.