Studies of soluble rat liver mitochondrial acid ATPases: II. Structural and immunological properties of ATPase 1

We described previously the existence of a soluble ATPase activity in rat liver mitochondria [1]. The purification and catalytic properties have been described [2]. In a continuation of these experiments, we have studied the immunologic and structural properties of one molecular form of this enzyme: ATPase I.We have prepared the antiserum anti-ATPase I and demonstrated the purity of our enzyme preparation by immunodiffusion and immunoelectrophoresis. An immunohistochemical method also confirmed the localization of ATPase I in the soluble fraction of mitochondria.The molecular weight of ATPase I was measured by G 100 Sephadex gel filtration and was found to be 18,400; electrophoresis on polyacrylamide gels gave a value of 18,600. The pHi of ATPase I was found to be 7,2.Amino acid analysis showed high amounts of aspartic acid, glutamic acid, serine and glycine. The molecular weight calculated from the total amino acid residues was found to be 17,000.Alanine is the NH2 terminal amino acid.The peptide maps obtained after degrading ATPase I with cyanogen bromide or trypsin are in accordance with the methionine, lysine and arginine residues we found in the ATPase I molecule.ATPase I does not appear to be a glycoprotein.     

Purification and Properties of a Hydrogenase from Desulfovibrio vulgaris

The soluble hydrogenase of Desulfovibrio vulgaris was purified and some of its properties are described. The molecular weight was determined for the enzyme by sedimentation equilibrium (45,400) and amino acid analysis (44,800). The hydrogenase appears to be a loosely coiled molecule or to have a high axial ratio, which is reflected in an unusually low sedimentation coefficient (2.58S) and a low diffusion coefficient (D 5.85). The molecular weight of the hydrogenase (41,000), as calculated by the Svedberg equation, was in general agreement with the sedimentation equilibrium molecular weight. Amino acid analysis revealed the presence of six halfcystine residues per mole of enzyme and a total of 417 amino acid residues. The specificity of the hydrogenase and its capacity to reduce certain low potential dyes and cytochrome c(3) was studied. Metal analysis of the hydrogenase indicated the presence of 1 mole of ferrous iron per mole of enzyme.     

Cloning and sequencing of the endo-cellulase cDNA from Robillarda sp. Y-20

An endo-cellulase cDNA has been screened from a Robillarda sp. Y-20 cDNA expression library using polyclonal antibodies (immunoscreening). Western blot analysis showed that recombinant CMCase I fused to beta-galactosidase with the molecular weight of approximately 150,000 was expressed in Escherichia coli Y1090. Sequence analysis of the cloned cDNA revealed that it consisted of 1,136 nucleotides. By comparison of the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of the purified protein (residues 1 to 18, determined by protein sequencing), the cDNA was found to lack 44 nucleotides at its 5' end corresponding to residues 1 to 15. Therefore, the mature cellulase (CMCase I) was deduced to be composed of 375 amino acid residues and the molecular weight of its protein was calculated to be 41,004. Yaguchi et al. reported that the N-terminal amino acid sequence of an endo-beta-1,4-glucanase (endo-cellulase) from Schizophyllum commune was homologous with the active site of various hen egg-white type lysozymes, and the homology offered evidence for a lysozyme-type mechanism in enzymatic hydrolysis of cellulase [Biochem. Biophys. Res. Commun. (1983) 116, 408-411]. Pentill? et al. also pointed out that some amino acid homology was found between endo-glucanase I from Trichoderma reesei and the lysozyme of the phage T4 [Gene (1986) 45, 253-263]. From the result of sequence alignment of the endo-cellulase from Robillarda sp. Y-20 and four kinds of lysozymes, there was a possibility that the endo-cellulase from Robillarda sp. Y-20 also hydrolyzes carboxymethyl cellulose by a lysozyme-type mechanism.     

Molecular weight,amino acid composition and other properties of membrane-bound ATPase fromBacillus megaterium KM

The Ca²⁺-activated ATPase fromBacillus megaterium KM has a molecular weight of 379000 as determined by sedimentation equilibrium in the analytical ultracentrifuge and 410000 as determined from sedimentation and diffusion coefficients. These values compare closely with molecular weights estimated for similar ATPases fromStreptococcus faecalis and mitochondria. On polyacrylamide gel electrophoresis in sodium dodecylsulfate two classes of subunit of molecular weight 68000 and 65000 are seen. They seem to be present in approximately equal proportion. The amino acid analysis gives a minimum molecular weight of 6250 and the amino acid composition is extremely close to that ofS. faecalis. The enzyme is denatured at 55°C and insensitive to oligomycin or ruthenium red in either the membrane-bound or soluble forms. The ATPase is estimated to comprise approximately 1% of the total cytoplasmic membrane protein.     

Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of phosphatidylcholine for enzymatic activity. Purification of the enzyme to homogeneity from bovine heart mitochondria was described more than a decade ago [H. G. Bock and S. Fleischer (1975) J. Biol. Chem. 250, 5774-5781]. We have modified the purification procedure so that it is faster, the yield has been improved, and the specific activity is greater by approximately 50%. The updated procedure has also been applied to isolate the enzyme from rat liver mitochondria. Characteristics of the enzyme from bovine heart and rat liver mitochondria have been compared and found to be similar with respect to: (1) purification characteristics; (2) amino acid composition; (3) pH optimum for enzymatic activity; (4) kinetic characteristics; (5) molecular weight as determined by sedimentation equilibrium in guanidine hydrochloride; (6) peptide maps; (7) immunological cross-reactivity. These studies show that 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria, though similar, are not identical.     

The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Identification and isolation.

Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor-modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.     

ATPase inhibitor from yeast mitochondria. Purification and properties.

1. Mitochondria from Candida utilis CBS 1516 and Sacchromyces cerevisiae JB 65 possess an ATPase-inhibitor activity. The inhibitor activity depends on the growth conditions of the yeast cells. It is markedly decreased when the cells are grown in the presence of a high concentration of glucose, which suggests that glucose represses the synthesis of the ATPase inhibitor or of a protein required for the insertion of the inhibitor into the inner mitochondrial membrane. 2. The ATPase inhibitor has been isolated from D. utilis mitochondria and purified to homogeneity. The minimal molecular weight calculated from amino acid composition is close to 7500. Dtermination of the molecular weight by sokium dodecylsulfate-polyacrylamide gel electrophoresis gives a value close to 6000. 3. The ATPas inhibitor of C. utilis mitochondria differs from the beef heart ATPase inhibitor by a number of properties. It has a lower molecular weight (6000-7500 vs 10500), a different amino acid composition, and a more acidic isoelectric point 5, 6 vs 7, 6). In spite of these differences, the C. utilis inhibitor cross-reacts with the ATPase of beef heart submitochondrial inhibitor-depleted particles. 4. The interaction of the C. utilis inhibitor with the ATPase of inhibitor-depleted particles requires the addition of Mg-2+-ATP or ATP in the incubation medium. 5. 14-C labelling of the C.utilis inhibitor has been achieved by growing C. utilis in a medium supplemented with [14-C]leucine. It has been found by titration experiments that the C. utilis 14-C-labelled inhibitor binds to the homologous submitochondrial inhibitor-depleted particles with a KD of about 10- minus 7 M. The number of binding sites is of the order of 0.1 nmol/mg protein.     

Purification and characterization of DNA-dependent ATPase II from Escherichia coli.

A new DNA-dependent ATPase was isolated and purified from soluble extracts of Escherichia coli. This enzyme, called ATPase II, has a molecular weight of 86,000 and exists in a monomeric state. It degrades ATP (or dATP) to ADP (or dADP) and Pi in the presence of magnesium and requires a double-stranded polynucleotide as cofactor. A correlation between the efficiency as cofactor and the melting point of the polynucleotide has been found; the lower the melting temperature, the higher the stimulation of ATPase II. The enzyme binds to single-stranded DNA and poly[d(A-T)] copolymer, but not to the double-stranded circular DNA (Form I) of simian virus 40.     

Human genetically polymorphic deoxyribonuclease: purification, characterization, and multiplicity of urine deoxyribonuclease I

A deoxyribonuclease I was purified from the urine of a 46-year-old male (a single individual) by using a series of column chromatographies to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to be a glycoprotein, containing 1 fucose, 7 galactose, 10 mannose, 6 glucosamine, and 2 sialic acid residues per molecule. The N-terminal amino acid sequence up to the 27th residue of the enzyme was similar to that of pancreatic deoxyribonuclease I from bovine and other species. The catalytic properties of the enzyme derived from a single individual closely resembled those of deoxyribonuclease I purified from human urine collected from several volunteers [Ito, K. et al. (1984) J. Biochem. 95, 1399-1406]. The purified enzyme was found to consist of multiple forms with different pI values. These findings are compatible with the existence of genetic polymorphism of deoxyribonuclease I in human urine previously reported [Kishi, K. et al. (1989) Hum. Genet. 81, 295-297]. This multiplicity of the urine enzyme might be due to variations in the primary structure and/or differences in the content of sialic acid.     

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolytic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction [Deutscher, J., & Saier, M. H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. The site of ATP-dependent phosphorylation in HPr of S. faecalis has now been determined. [32P]P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, we obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, we isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a new ribonuclease from Aspergillus saitoi.

From a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12,500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38,000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electrophoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70 degrees for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid is greater than adenylic acid is greater than cytidylic acid is greater than uridylic acid.     

Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1

Catechol 1,2-dioxygenase (pyrocatechase) has been purified to homogeneity from Pseudomonas putida mt-2. Most properties of this enzyme, such as the absorption spectrum, iron content, pH stability, pH optimum, substrate specificity, Km values, and amino acid composition, were similar to those of catechol 1,2-dioxygenase obtained from Pseudomonas arvilla C-1 [Y. Kojima et al. (1967) J. Biol. Chem. 242, 3270-3278]. These two catechol 1,2-dioxygenases were also found, from the results of Ouchterlony double diffusion, to share several antigenic determinants. The molecular weight of the putida enzyme was estimated to be 66,000 and 64,000 by sedimentation equilibrium analysis and Sephadex G-200 gel filtration, respectively. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to Mr 32,000. The NH2-terminal sequence, which started with threonine, was determined up to 30 residues by Edman degradation. During the degradation, a single amino acid was released at each step. The NH2-terminal sequence up to 20 residues was identical to that of the beta subunit of the arvilla enzyme, with one exception at step 16, at which arginine was observed instead of glutamine. The COOH-terminal residue was deduced to be arginine on carboxypeptidase A and B digestions and on hydrazinolysis. These results indicate that the putida enzyme consists of two identical subunits, in contrast to the arvilla enzyme which consists of two nonidentical subunits, alpha and beta [C. Nakai et al. (1979) Arch. Biochem. Biophys. 195, 12-22], although these two enzymes have very similar properties.     

Mitochondrial ATPase complex of Aspergillus nidulans and the dicyclohexylcarbodiimide-binding protein.

The dicyclohexylcarbodiimide-binding protein of Aspergillus nidulans has been identified as the smallest subunit of the mitochondrial ATPase complex, and has a molecular weight of approximately 8000. It is extractable from whole mitochondria and from the purified enzyme in neutral chloroform/methanol, contains 30% polar amino acids, and the N-terminal amino acid has been identified as tyrosine. Using a double-labelling technique in the absence and presence of cycloheximide, followed by immunoprecipitation of the enzyme complex with antiserum against Neuospora crassa F1 ATPase, it has been shown that this subunit is synthesized on cytoplasmic ribosomes.     

The Escherichia coli pldC gene encoding lysophospholipase L(1) is identical to the apeA and tesA genes encoding protease I and thioesterase I, respectively.

We deduced the amino acid sequence of Escherichia coli lysophospholipase L(1) by determining the nucleotide sequence of the pldC gene encoding this enzyme. The translated protein was found to contain 208 amino acid residues with a hydrophobic leader sequence of 26 amino acid residues. The molecular weight of the purified enzyme (20,500) was in good agreement with the predicted size (20,399) of the processed protein. A search involving a data bank showed that the nucleotide sequence of the pldC gene was identical to those of the apeA and tesA genes encoding protease I and thioesterase I, respectively. Consistent with the identity of the pldC gene with these two genes, the enzyme purified from E. coli overexpressing the pldC gene showed both protease I and thioesterase I activities.     

Acetylation of prostaglandin synthetase by aspirin. Purification and properties of the acetylated protein from sheep vesicular gland.

We previously presented evidence that aspirin (acetylsalicylic acid) inhibits prostaglandin synthetase by acetylating and active site of the enzyme. In the current work, we have labeled the enzyme from an aceton-pentane powder of sheep vesicular gland using [acetyl-3H]aspirin and purified the [3H]acetyl-protein to near homogeneity. The final preparation contains protein of a single molecular weight (85 000) and an amino-terminal sequence of Asp-Ala-Gly-Arg-Ala. The [3H]acetyl-protein contained 0.5 mol of acetyl residues per mol of protein based on amino acid composition but only a single sequence was found.     

Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme. Effects of flavonoids.

Soluble ATPase (F1) has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2, and 5.4. Kinetic properties (Vm and Km (atp) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.

A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.     

The calcium-binding ATPase inhibitor protein from bovine heart mitochondria. Purification and properties

Two ATPase inhibitor proteins were isolated together from bovine heart mitochondria by a new procedure; each was purified further. The one inhibitor is a Ca2+-binding protein. It was found to contain 2 cysteine residues/mol as well as threonine and proline residues, all of which the other inhibitor (first isolated by Pullman and Monroy (Pullman, M.E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769] lacks. Its minimal molecular weight was 6390 with 62 amino acid residues/mol, and its isoelectric point was 4.6. Besides differences in size, composition, and response to Ca2+, the two inhibitor proteins also differed in response to sulfhydryl compounds, pH, KCl, and cardiolipin. Inhibition by the two inhibitor proteins was additive. Both cross-reacted with mitochondrial ATPase from rat skeletal muscle. Calmodulin, with or without Ca2+, had no effect on the activity of either inhibitor protein. Antibody to the Ca2+-binding inhibitor protein did not interact with the Pullman-Monroy inhibitor or have any effect on its activity. The antibody interacted with intact submitochondrial particles that contained both inhibitor proteins but not with particles from which only the Ca2+-binding inhibitor had been removed. Clearly, the two inhibitors are distinct immunologically as well as in other properties. The two types of inhibitor protein were also isolated from rat skeletal muscle mitochondria by the new procedure.     

Properties of mitochondrial adenosine triphosphatase isolated from rat liver.

Soluble, oligomysin-insensitive ATPase was isolated from liver mitochondria by a new technique [Drahota and Houst?k 1977]. Study of the resultant enzyme preparation provided further evidence that the isolated protein displays properties typical of mitochondrial ATPase (specific activity about 100 U/mg, optimum pH 8.2, activation by various bivalent cations and reassociation with membranes deprived of ATPase).