Further characterization of L-β-hydroxyacid dehydrogenase from Drosophila

L-β-Hydroxyacid dehydrogenase (L-β-hydroxyacid--NAD-oxidoreductase, EC 1.1.1.45) of Drosophila is composed of two, identical subunits with a molecular weight of approx. 33 300. The enzyme was purified 938-fold from Drosophila melanogaster. An isoelectric point of 8.6 was determined for L-β-hydroxyacid dehydrogenase. An amino acid analysis was conducted of the purified enzyme. A single subunit was obtained by SDS-gel electrophoresis of the purified enzyme. Translation of larval and adult mRNA in a mRNA-dependent reticulocyte lysate, followed by immune precipitation using anti-L-β-hydroxyacid dehydrogenase IgG revealed a single L-β-hydroxyacid dehydrogenase subunit of 33 300. Larval and adult proteins were the same size. The enzyme does not appear to be subjected to substantial post-translational modifications.     

Enzyme instability and proteolysis during the purification of an alcohol dehydrogenase from Drosophila melanogaster.

The alcohol dehydrogenase of the Drosophila melanogaster adhUF allele (alloenzyme with ultra-fast electrophoretic mobility) was unstable in crude or partially purified preparations. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that inactivation was porbably due to proteolytic degradation, and new method of purification of the enzyme was developed. After three steps, namely salmine sulphate precipitation, hydroxyapatite chromatography and Sephadex G-100 gel filtration, a 10-fold purified preparation was obtained. The enzyme produced was relatively stable compared with alcohol dehydrogenase purified by other methods, and was shown to be proteinase-free. The enzyme had a subunit mol.wt. of 24000 and had a single thiol residue per subunit available for titration with 5,5'-dithiobis-(2-nitrobenzoic acid). The amino acid composition and C-terminal amino acid sequence of the enzyme were determined. The substrate specificity of this alcohol dehydrogenase was also characterized. These results are discussed in relation to experiments on the evolutionary significance of thermostability at the adh locus.     

Developmentally regulated alternate modes of expression of the Gpdh locus of Drosophila.

Immunoblot analyses have been performed on extracts prepared from Drosophila melanogaster. Those analyses have revealed two subunit forms of enzyme glycerol 3-phosphate dehydrogenase (GPDH) in larval tissues and in adult abdominal tissues. Thoracic tissue, which accounts for the bulk of the adult GPDH, has only one subunit form, the smaller. The two subunit forms differ by approximately 2400 daltons. In agreement with previous genetic and biochemical data indicating that this enzyme is encoded by a single structural gene, analyses of extracts prepared from a strain carrying a GPDH null mutation detect no GPDH polypeptides in larvae or adults. Similarly, analyses of extracts prepared from a strain carrying a mutation which produces a GPDH polypeptide that differs in size from wild-type reveal a change in the adult thoracic GPDH polypeptide as well as a change in both GPDH polypeptides found in larvae. Total Drosophila RNA prepared from larvae or newly eclosed adults has been translated in a mRNA-dependent cell-free system. GDPH was immunoprecipitated from the translation products and analyzed. Two subunit forms of GPDH were immunoprecipitated from translation products whose synthesis was directed by larval RNA and only one was detected in the polypeptides synthesized from adult RNA. The GPDH polypeptides synthesized in vitro are approximately the same size as the corresponding polypeptides found in vivo. The relative proportion of total GPDH represented by each subunit form synthesized in vitro is similar to those found in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of revertants of lys2 and lys5 mutants as well as alpha-aminoadipate-semialdehyde dehydrogenase from Saccharomyces cerevisiae

alpha-Aminoadipate-semialdehyde dehydrogenase catalyzes the conversion of alpha-aminoadipate to alpha-aminoadipate-semialdehyde in the biosynthetic pathway of lysine in yeasts and molds. Mutants belonging to lys2 and lys5 loci of Saccharomyces cerevisiae lacked the alpha-aminoadipate-semialdehyde dehydrogenase activity. Complementation in vitro was demonstrated by combining the extracts from different lys2 and lys5 mutants. Some of the revertants of lys2 and lys5 mutants exhibited lower specific activity and higher thermolability of alpha-aminoadipate-semialdehyde dehydrogenase than the enzyme from wild-type cells. The enzyme was partially purified from wild-type cells and the molecular weight of the enzyme was estimated on a Sephacryl S-300 column at 180,000. Results from the revertant analysis and in vitro complementation indicated LYS2 and LYS5 as structural genes, each encoding a subunit of this large enzyme.     

Expression and purification of the alpha and beta subunits of Drosophila casein kinase II using a baculovirus vector.

Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.     

Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase

An NAD(+)-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase has been purified to homogeneity from the yeast Saccharomyces cerevisiae. The purified enzyme exhibits a final specific activity of 5.4 units mg-1 and is represented by a single protein of apparent Mr = 33,000-38,000 as determined by sodium dodecyl sulfate gel electrophoresis. A native Mr = 64,000 was determined by gel filtration, suggesting a homodimer subunit structure. Cross-linking experiments with dimethyl suberimidate confirmed the dimeric structure. The enzyme is specific for NAD+ and is not dependent on Mg2+ for activity. The forward reaction initial velocity kinetics are consistent with a sequential reaction mechanism. With this model, Km values for NAD+ and (6R,S)-5,10-methylene-THF are 1.6 and 0.06 mM, respectively. In contrast to all other previously described eukaryotic 5,10-methylene-THF dehydrogenases, the purified enzyme is apparently monofunctional, with undetectable 5,10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities. Subcellular fractionation of yeast indicates the enzyme is cytoplasmic, with no NAD(+)-dependent 5,10-methylene-THF dehydrogenase detectable in mitochondria. The activity was found in all yeast strains examined, at all stages of growth from the lag phase through the stationary phase.     

An unstable small-colony variant of a noninvasive mutant of Salmonella typhimurium is highly invasive for MDCK cells

Abstract A sorbitol dehydrogenase was purified from the membrane fraction of Gluconobacter suboxydans KCTC 2111 (= ATCC 621) by chromatography on CM-, DEAE-, Mono S and Superose 12 columns. The purified enzyme showed a single activity band upon nondenaturing polyacrylamide gel electrophoresis (PAGE) and three subunits of 75, 50 and 14 kDa upon SDS-PAGE. When purified preparations of the enzyme were reconstituted with pyrroloquinoline quinone (PQQ), the specific enzyme activity was significantly increased (up to 9-fold). The absorption spectrum of purified sorbitol dehydrogenase in the reduced state exhibited three absorption maxima (417, 522 and 552 nm) which is in accordance with the typical absorption spectrum of cytochrome c . The 50 kDa subunit appeared as a red band on unstained SDS-gels suggesting its identity as a cytochrome. Fluorescence spectra of extracts from purified sorbitol dehydrogenase showed an excitation maximum at 370 nm and an emission maximum at 465 nm, which conformed to those of authentic PQQ. The purified enzyme showed a rather broad substrate specificity with significant activity toward D-mannitol (68%) and D-ribitol (70%) as well as D-sorbitol (100%). The PQQ-dependent sorbitol dehydrogenase described in this study is clearly different from the FAD-dependent sorbitol dehydrogenase from G. suboxydans var. α IFO 3254 strain in its cofactor requirement and substrate specificity.     

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

An NAD(P)-dependent glutamate dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme is a hexamer (subunit mass 45 kDa) which dissociates into lower states of association when submitted to gel filtration. Isoelectric focusing analysis of the purified enzyme showed a pI of 5.7 and occasionally revealed microheterogeneity. The enzyme is strictly specific for the natural substrates 2-oxoglutarate and L-glutamate, but is active with both NADH and NADPH. S. solfataricus glutamate dehydrogenase revealed a high degree of thermal stability (at 80 C the half-life was 15 h) which was strictly dependent on the protein concentration. Very high levels of glutamate dehydrogenase were found in this archaebacterium which suggests that the conversion of 2-oxoglutarate and ammonia to glutamate is of central importance to the nitrogen metabolism in this bacterium.     

The purification and characterization of glutaryl-coenzyme A dehydrogenase from porcine and human liver

Glutaryl-CoA dehydrogenase, a multifunctional enzyme responsible for dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA, has been purified 1,680-fold from porcine liver mitochondria. The purified porcine enzyme has a subunit molecular weight of 47,800 and a native molecular weight of 190,500. Porcine glutaryl-CoA dehydrogenase catalyzed the conversion of [1,5-14C]glutaryl-CoA to [14C] crotonyl-CoA and 14CO2 in a 1:1:1 ratio. The porcine enzyme has Km values for electron transfer flavoprotein and glutaryl-CoA of 1.1 and 3.3 microM, respectively, and turnover numbers of 860 mol of electron transfer flavoprotein/min/mol of glutaryl-CoA dehydrogenase and 327 mol of glutaryl-CoA/min/mol of glutaryl-CoA dehydrogenase. Human glutaryl-CoA dehydrogenase has been purified 1,278-fold from human liver mitochondria. The purified human enzyme has a subunit molecular weight of 58,800 and a native molecular weight of 256,000. Human glutaryl-CoA dehydrogenase showed a reaction of only partial identity when compared to porcine glutaryl-CoA dehydrogenase by Ouchterlony double immunodiffusion analysis using antiserum raised against and monospecific for porcine glutaryl-CoA dehydrogenase.     

Baculovirus expression reconstitutes Drosophila mitochondrial DNA polymerase.

Drosophila mitochondrial DNA polymerase has been reconstituted and purified from baculovirus-infected insect cells. Baculoviruses encoding full-length and mature forms of the catalytic and accessory subunits were generated and used in single and co-infection studies. Recombinant heterodimeric holoenzyme was reconstituted in both the mitochondria and cytoplasm of Sf9 cells and required the mitochondrial presequences in both subunits. The recombinant holoenzyme contains DNA polymerase and 3'-5' exonuclease that are stimulated substantially by both salt and mitochondrial single-stranded DNA-binding protein. Thus, the recombinant enzyme exhibits biochemical properties indistinguishable from those of the native enzyme from Drosophila embryos. Production of the catalytic subunit alone yielded soluble protein with the chromatographic properties of the heterodimeric holoenzyme. However, the purified catalytic core has a 50-fold lower specific activity. This provides evidence of a critical role for the accessory subunit in the catalytic efficiency of Drosophila mitochondrial DNA polymerase.     

Purification of a lysosomal DNase from Drosophila melanogaster.

An acid DNase was purified from Drosophila melanogaster till apparent homogeneity by six consecutive chromatographic steps. The enzyme is a lysosomal DNase, because it is glycosylated and carries 1.8-2.4 mol of mannose-6-phosphate/mol of enzyme. The enzyme is fully active without any divalent cation and introduces single stranded nicks into a supercoiled DNA.     

Malonyl-coenzyme A reductase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a new autotrophic CO(2) fixation pathway in Chloroflexus aurantiacus and some archaebacteria. The initial step is acetyl-coenzyme A (CoA) carboxylation to malonyl-CoA by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate. This reduction step was studied in Chloroflexus aurantiacus. A new enzyme was purified, malonyl-CoA reductase, which catalyzed the two-step reduction malonyl-CoA + NADPH + H(+) --> malonate semialdehyde + NADP(+) + CoA and malonate semialdehyde + NADPH + H(+) --> 3-hydroxypropionate + NADP(+). The bifunctional enzyme (aldehyde dehydrogenase and alcohol dehydrogenase) had a native molecular mass of 300 kDa and consisted of a single large subunit of 145 kDa, suggesting an alpha(2) composition. The N-terminal amino acid sequence was determined, and the incomplete gene was identified in the genome database. Obviously, the enzyme consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain. No indication of the presence of a prosthetic group was obtained; Mg(2+) and Fe(2+) stimulated and EDTA inhibited activity. The enzyme was highly specific for its substrates, with apparent K(m) values of 30 microM malonyl-CoA and 25 microM NADPH and a turnover number of 25 s(-1) subunit(-1). The specific activity in autotrophically grown cells was 0.08 micromol of malonyl-CoA reduced min(-1) (mg of protein)(-1), compared to 0.03 micromol min(-1) (mg of protein)(-1) in heterotrophically grown cells, indicating downregulation under heterotrophic conditions. Malonyl-CoA reductase is not required in any other known pathway and therefore can be taken as a characteristic enzyme of the 3-hydroxypropionate cycle. Furthermore, the enzyme may be useful for production of 3-hydroxypropionate and for a coupled spectrophotometric assay for activity screening of acetyl-CoA carboxylase, a target enzyme of potent herbicides.     

Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody

Betaine-aldehyde dehydrogenase was purified from spinach leaves and characterized. The molecular weight of the enzyme was estimated to be 120 kDa by a gel filtration chromatography. The enzyme was judged to consist of two identical pieces of the monomeric subunit with molecular weight of 60 kDa. A specific polyclonal antibody was raised against the enzyme subunit.     

Succinic semialdehyde dehydrogenase from mammalian brain: subunit analysis using polyclonal antiserum.

1. NAD(+)-dependent succinic semialdehyde dehydrogenase was purified to apparent homogeneity from rat brain and highly purified from human brain. 2. Molecular exclusion chromatography of the purified enzymes on Sephadex G-150 and G-200 revealed M(r) values of 203,000 and 191,000 for rat and human, respectively. 3. Electrophoresis on sodium dodecylsulfate polyacrylamide gels revealed a single subunit of M(r) 54,000 for rat and 58,000 for human. Isoelectric focusing of the purified rat enzyme yielded a pI of 6.1. 4. For both proteins, Km values for short-chain aldehydes acetaldehyde and propionaldehyde ranged from 0.33 to 2.5 mM; Km values for succinic semialdehyde were in the 2-4 microM range. 5. The subunit structure of both enzymes was investigated in brain extracts and purified preparations by immunoblotting, using a polyclonal rabbit antiserum against the purified rat brain enzyme. 6. For rat and human extracts, single bands were detected at M(r) 54,000 and 58,000, comparable to findings in the purified preparations. Immunoblotting analyses in other species (guinea pig, hamster, mouse and rabbit) revealed single subunits of M(r) 54,000-56,500.     

Regulation of rat liver NADP+-isocitrate dehydrogenase during aging

In an attempt to understand the mechanism of aging in relation to the differences in enzyme regulation, the induction and kinetic properties of NADP⁺ -isocitrate dehydrogenase of the liver of immature (6 weeks), mature (13 weeks), adult (33 weeks) and old (85 weeks) female rats were studied. The specific activity of the cytoplasmic and mitochondrial NADP+ -isocitrate dehydrogenase increased up to the adult age (33 weeks) and decreased in the old rats (85 weeks). Overiectomy decreased and estradiol administration induced activity of both the mitochondrial and eytoplasmic enzyme in the liver ol immature, mature and adult rats but had no significant effect in old rats. However, the activity of mitochondrial NADP⁺ -isocitrate dehydrogenase decreased and eytoplasmic NADP+ -isocitrate dehydrogenase increased following ovariectomy in old rats (85 weeks). Hormone-mediated induction of enzyme activity was actinomycin D sensitive. The Km for isocitrate and NADP, Ki value for oxalomalate, heat stability and electrophoretic mobility of the purified enzyme from the cytosol fraction of the liver of immature and old rats were similar. It can he concluded that the enzyme does not change structurally with age. Part of this work was presented at the 48th Annual General Meeting of the Society of Biological Chemist, India, 1979.     

Purification of mitochondrial NADH dehydrogenase from Drosophila hydei and comparison with the 'heat-shock' polypeptides.

Mitochondrial NADH dehydrogenase (NADH:(acceptor) oxidoreductase, EC .6.99.3) from either Drosophila hydei larvae or embryos has been purified 150- and 120-fold, respectively. The purified enzyme appeared homogeneous and showed a molecular weight of 57 000. The molecular weight of the nondenatured enzyme was 79 000. On isoelectro-focussing of the preparation, two fractions were observed, a major one with an isoelectric point of 6.2 and a minor fraction with an isoelectric point of 4.9. Straight-line kinetics in Lineweaver-Burk plots were observed for the purified enzyme with a Km of 0.040 mM. The Km was not changed during the purification procedure, suggesting that the enzyme was not denatured or inactivated. The pH optimum of the purified enzyme was 5.6. The molecular weight of the purified mitochondrial NADH dehydrogenase does not correspond to that of one of the 'heat-shock' polypeptides.     

Purification and Characterization of Sorbitol Dehydrogenase from Bovine Brain

Sorbitol dehydrogenase (EC 1.1.1.14) was isolated from bovine brain and purified 3,000-fold to apparent homogeneity, as judged by polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 36 units/mg of protein; a molecular weight of 39,000 for each of the four identical subunits and 155,000 for the intact enzyme were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography, respectively. The presence of one Zn2+ per subunit was confirmed by atom absorption spectroscopy; inactivation of the enzyme by metal-chelating agents points to the essential role that Zn2+ plays in the catalytically competent enzyme. The enzyme is also inactivated by thiol-blocking reagents; with respect to inactivation by sodium pyrophosphate, sorbitol dehydrogenase is different from closely related alcohol dehydrogenase.     

Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast

The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.     

Partial purification and characterization of a pyruvate dehydrogenase-complex-inactivating enzyme from rat liver.

An enzyme inactivating the pyruvate dehydrogenase complex (inactivase) was purified about 8000-fold from rat liver by differential centrifugation, acid extraction of a lysosomerich 25000 g pellet, acetone fractionation, and adsorption on calcium phosphate gel. By exclusion chromatography on Sephadex G-100 a molecular weight of 21 000 was estimated. The purified enzyme was most stable at pH 5.8 in potassium phosphate buffer, and at pH 4.5 in McIlvaine buffer. At high dilutions the enzyme was very labile and was remarkably stabilized by high salt concentrations. Enzyme activity is inhibited by native rat blood serum, iodoacetamide and leupeptin, but not by phenylmethanesulphonyl fluoride, suggesting that it belongs to the class of thiol proteinases. Among various enzymes tested, only 2-oxoglutarate dehydrogenase was attacked by the inactivase to a similar extent to the pyruvate dehydrogenase complex. Studies on the inactivation mechanism indicate that although the overall reaction is completely lost after treatment with inactivase, each individual step of the multienzyme complex retains full catalytic activity. As judged from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the transacetylase subunit appears to be degraded into several smaller fractions.     

Solubilization, purification and characterization of D-alanine dehydrogenase from Pseudomonas aeruginosa and effects of solubilization on its properties

D-alanine dehydrogenase, an inducible, membrane associated enzyme of Pseudomonas aeruginosa was solubilized from envelope preparations by treatment with Triton X-100 and purified 31-fold in the presence of 0.05% Triton X-100 to 60% homogeneity. Gel electrophoresis indicated the presence of a single subunit of approximately 49,000 molecular weight. The enzyme contained FAD, and absorption spectra were typical of an iron-sulfur flavoprotein. Solubilization produced significant changes in some properties of the enzyme: solubilized enzyme showed increased affinity for D-alanine; a broader substrate specificity; and increased temperature sensitivity, compared with the membrane associated form.