Purification and properties of Bacillus subtilis aspartate transcarbamylase.

Aspartate transcarbamylase from Bacillus subtilis has been purified to apparent homogeneity. A subunit molecular weight of 33,500 +/- 1,000 was obtained from electrophoresis in polyarcylamide gels containing sodium dodecyl sulfate and from sedimentation equilibrium analysis of the protein dissolved in 6 M guanidine hydrochloride. The molecular weight of the native enzyme was determined to be 102,000 +/- 2,000 by sedimentation velocity and sedimentation equilibrium analysis. Aspartate transcarbamylase thus appears to be a trimeric protein; cross-linking with dimethyl suberimidate and electrophoretic analysis confirmed this structure. B. subtilis aspartate transcarbamylase has an amino acid composition quite similar to that of the catalytic subunit from Escherichia coli aspartate transcarbamylase; only the content of four amino acids is substantially different. The denaturated enzyme has one free sulfhydryl group. Aspartate transcarbamylase exhibited Michaelis-Menten kinetics and was neither inhibited nor activated by nucleotides. Several anions stimulated activity 2- to 5-fold. Immunochemical studies indicated very little similarity between B. subtilis and E. coli aspartate transcarbamylase or E. coli aspartate transcarbamylase catalytic subunit.     

Disruption of the quaternary structure of (Na+ + K+)-dependent adenosine triphosphatase by Triton X-100.

(Na⁺ + K⁺)-dependent adenosine triphosphatase (NaK ATPase) consists of two polypeptide chains, a large polypeptide with a molecular weight of about 100,000, and a sialoglycoprotein with a molecular weight of about 40,000. In the presence of Triton X-100 both polypeptides react to form high molecular weight aggregates with apparent molecular weights of 168,000, 200,000 and 260,000. These aggregates arise as a result of disulfide bond formation which results from the autooxidation of sulfhydryl groups on the two polypeptides of NaK ATPase. These data are discussed in light of studies aimed at determining the size and subunit structure of membrane proteins with Triton X-100.     

Localization of the substrate and oxalacetate binding site of succinate dehydrogenase.

Succinate dehydrogenase is composed of two subunits, one of molecular weight 70,000, containing FAD in covalent linkage to a histidyl residue of the polypeptide chain, the other subunit of molecular weight 30,000. The fact that substrate, substrate analogs, and oxalacetate prevent inactivation of the enzyme by thiol-specific agents indicates that a thiol group must be present in close proximity to the flavin. Comparison of the incorporation of radioactivity into each subunit in the presence and absence of succinate or malonate shows that both substrate and competitive inhibitors protect a sulfhydryl group of the 70,000-molecular weight subunit. This indicates that a thiol group of the flavoprotein subunit is part of the active site. Similar investigations using oxalacetate as a protecting agent indicate that the tight binding of oxalacetate to the deactivated enzyme also occurs in the flavoprotein subunit, and may involve the same thiol group which is protected by succinate from alkylation by N-ethylmaleimide. It is clear, therefore, that not only the flavin site but also an essential thiol residue are located in the 70,000-molecular weight subunit. A second thiol group, located in the 30,000-molecular weight subunit, also binds N-ethylmaleimide covalently under similar conditions, without being part of the active site. Succinate, malonate, and oxalacetate do not influence the binding of this inhibitor to the thiol group of the lower molecular weight subunit. Using maleimide derivatives of nitroxide-type spin labels, it has been possible to demonstrate the presence of two types of thiol groups in the enzyme which form covalent derivatives with the spin probe. When the enzyme is treated with an equimolar quantity of the spin probe, a largely isotropic electron spin resonance spectrum is obtained, indicating a high probe mobility. When this site is first blocked by treating the enzyme with an equimolar quantity of N-ethylmaleimide, followed by an equimolar amount of spin label, the label is strongly immobilized with a splitting of 64 gauss. It is suggested that the sulfhydryl group which is involved in the immobilized species is at the active site.     

Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases.

The 125- and 48-kDa subunits of bovine DNA polymerase delta have been isolated by SDS-polyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA polymerase delta preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116-kDa polypeptides were used to design primers for the polymerase chain reaction to determine the nucleotide sequence of a full-length cDNA encoding the catalytic subunit of bovine DNA polymerase delta. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123,707. This is in agreement with the molecular weight of 125,000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA polymerase delta with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA polymerase delta contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA polymerase delta, but these regions share little or no homology with the alpha polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA polymerases, whereas one region is not homologous to any other DNA polymerase that has been sequenced thus far.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.     

Molecular properties of yeast glucokinase

Summary Glucokinase from baker's yeast has been purified to homogeneity. The molecular weight of the subunit is 51,000. The native enzyme sediments with s_20,w values in the range of 19 to nearly 4 S. The presence of glucose and phosphate favors the heavier species while ATP causes depolymerization. Titration experiments with the Ellman reagent support this view.The enzyme subunit has four sulfhydryl residues of which one is more reactive than the other three. However, it does not seem to be directly responsible for the catalytic activity. The amino acid composition of the enzyme is similar to those of the hexokinases P1 and P2 but for aspartic acid and histidine.     

The structure of prekeratin

The subunit of epidermal prekeratin has been shown to consist of three polypeptide chains. One of these has a molecular weight of 72,000 and two have molecular weights of 60,000, giving a total subunit molecular weight of 192,000. The prekeratin molecule, examined by ultracentrifugation, appears monodisperse and has a molecular weight of about 375,000 which is twice the subunit weight. It is concluded that prekeratin consists of a pair of three-stranded subunits and that this dimer is most probably of physiological significance, being a building block of epidermal filaments (“α-keratin”).     

Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase.

The catalytic subunit of rabbit skeletal muscle cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been isolated in pure form. It has a molecular weight of 41,300, as determined by sedimentation equilibrium, which is in good agreement with the value of 41,000 determined by electrophoresis in the presence of sodium dodecyl sulfate. Sedimentation velocity determinations indicate that the subunit has an S20,w value of 3.12 which is essentially independent of protein concentration. These experiments are interpreted as indicating that the catalytic subunit dissociated from the holoenzyme exists as a monomer in solution. The least abundant amino acid is half-cystine, which was calculated to be present at 2.8 mol/mol of protein. The sulfhydryl reagents, N-ethylmaleimide, p-chloromercuribenzoic acid, and 5,5'-dithiobis(2-nitrobenzoic acid) inhibit the enzymatic activity of the subunit; inhibition by the two latter compounds can be reversed by 2-mercaptoethanol. Binding of 1 mol of N-ethylmaleimide/mol of protein results in almost complete inhibition. The isolated catalytic subunit contains 2.2 mol of tightly bound phosphate/mol of protein. Identification of either O-phosphoserine or O-phosphothreonine after partial acid hydrolysis indicates that at least part of the endogeneous phosphate exists as the phospho ester of one of these amino acids. Two peaks of catalytic activity corresponding to isoelectric points of pH 7.4 and 8.5 were identified by isoelectric focusing. Both forms utilize the same substrates and have similar sedimentation constants.     

Pyruvate carboxylase from a thermophilic Bacillus: some molecular characteristics.

Analysis of the native enzyme and of the subunits produced upon its denaturation shows that pyruvate carboxylase from a thermophilic Bacillus is a tetramer with a molecular weight (mean value) of 558,000 and that the four polypeptide subunits are probably identical. The three functions (carboxyl carrier, carboxylation, and carboxyl transfer) in the pyruvate carboxylation reaction must therefore reside in this quarter-molecular polypeptide. The enzyme molecule contains four atoms of zinc and four molecules of D-biotin, and in the electron microscope the disposition of its four subunits presents a rhombic appearance. Reaction of the denatured enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reveals 10 sulfhydryl groups/subunit. In the native enzyme less than one of these groups reacts with DTNB. By contrast, all of these groups (11/subunit) of the native chicken liver pyruvate carboxylase are accessible to DTNB. The thermophile enzyme is also more resistant to other sulfhydryl reagents and to denaturation under certain conditions than the avian enzyme.     

Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase isolated from Rhodopseudomonas sphaeroides 2.4.1.Ga was separated into two different forms by DEAE-cellulose column chromatography. Both forms, designated Peak I and Peak II have been purified to homogeneity by the criterion of polyacrylamide disc-gel electrophoresis. The Peak I carboxylase has a molecular weight of 550,000, while the Peak II carboxylase is a smaller protein having a molecular weight of approximately 360,000. Sodium dodecyl sulfate electrophoresis revealed a large subunit for both enzymes which migrates similarly to the large subunit of spinach Rbu-P2 carboxylase. The Peak I enzyme also exhibited a small subunit having a molecular weight of 11,000. No evidence for a smaller polypeptide was found associated with the Peak II enzyme. Antisera prepared against the Peak I enzyme inhibited Peak I enzymatic activity, but had no effect on the activity of the Peak II enzyme. The two enzymes exhibited marked differences in catalytic properties. The Peak I enzyme exhibits optimal activity at pH 8.0 and is inhibited by low concentrations of 6-phosphogluconate, while the Peak II enzyme has a pH optimum of 7.2 and is relatively insensitive to 6-phosphogluconate.     

Absence of accessory subunit in the DNA polymerase gamma purified from yeast mitochondria

In yeast and animals, replication of the mitochondrial genome is carried out by the DNA polymerase gamma. In mammals this polymerase is composed of a catalytic and an accessory subunit. Yeast DNA polymerase gamma was purified over 6600-fold from mitochondria. The catalytic polypeptide of this enzyme was identified as a 135-kDa protein by a photochemical crosslinking procedure and its native molecular weight was estimated between 120 and 140 kDa by gel filtration and glycerol gradient sedimentation. These results indicate that yeast DNA polymerase gamma contains only one subunit and thus has a different quaternary structure from its counterpart in animals.     

Purification and characterization of shikimate kinase enzyme activity in Bacillus subtilis.

In Bacillus subtilis shikimate kinase enzyme activity can be demonstrated when a small polypeptide forms a trifunctional complex with the bifunctional enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase-chorismate mutase. The shikimate kinase polypeptide whoch carries the catalytic site has been purified to homogeneity by a five-step procedure. The skikimate kinase was determined to have a molecular weight of 10,000 by superfine Sephadex G-75 thin layer chromatography and by calculation of the minimum chemical molecular weight from its amino acid composition. This number corresponds closely to the molecular weight determined by the mobility of the protein following electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The enzyme aggregates with itself forming larger molecular weight proteins. Thes aggregational pattersn depend on protein concentration and sulfhydryl bridges. The enzyme activity is completely inhibited by EDTA and the requirement for Mg2+ can be partially replaced by Mn2+, Ca2+, and Co2+. The inhibition of shikimate kinase activity by p-hydroxymercuribenzoate is reversed completely when the enzyme complex is treated with dithiothreitol, suggesting the sulfhydryl groups may be involved with the active site. The trifunctional complex is relatively unstable, and the nonidentical subunits dissociate readily. This dissociation results in a 99% loss in shikimate kinase activity and a 30% decrease in the chorismate mutase-DAHP synthetase activities. Shikimate kinase activity is subject to a variety of controls. It is inhibited by the allosteric effectors chorismate and prephenate, the products of the reaction, ADP, and shikimate 5-phosphate. The activity responds to changes in the energy charge of the cell. Because of the variety of controls exerted on this enzyme, this member of the regulatory complex may represent the key enzyme in the allosteric control of the synthesis of the common precursors of aromatic acid synthesis.     

Molecular weight of (Na+,K+)ATPase from shark rectal gland.

The (Na+,k+)ATPase from the rectal gland of Carcharhinus obscurus has been solubilized in Lubrol WX as an active complex containing 379,900 g of protein and 61 mol of phospholipid. This detergent-lipid-protein complex contains two catalytic subunits of molecular weight 106,400 and four glycopeptide subunits of protein molecular weight 36,600. The latter subunit has a total molecular weight of 51,700 when the carbohydrate is included. Attempts to dissociate this active enzyme complex to smaller size by increasing the detergent concentration led to inactivation. Thus, the smallest active particle in the presence of Lubrol WX contains the two polypeptide subunits in a mole ratio of 2:4 under conditions where the micellar form of detergent is present at a 70:1 molar ratio. This large excess of Lubrol WX eliminates any possibility of artificial togetherness as the result of statistical considerations.     

Partial Purification and Some Properties of the Staphylococcus aureus Cytoplasmic Nitrate Reductase

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.     

Regulatory subunit of cyclic AMP-dependent protein kinase I from porcine skeletal muscle: purification and proteolysis.

The regulatory subunit of cyclic AMP-dependent protein kinase I was purified to homogeneity from porcine skeletal muscle by two different procedures, one relying on affinity chromatography with cyclic AMP-Sepharose and the other relying exclusively on ion-exchange and molecular seive chromatography. Both procedures were adapted so that catalytic subunit also could be purified from the same enzyme preparation. In its native form the regulatory subunit was a dimer having a molecular weight of 92,500. Polyacrylamide gels run under denaturing conditions indicated that the dimer was composed of two identical subunits having a molecular weight 45,500. In addition to the dimeric regulatory subunit, a second, smaller cyclic AMP-binding protein frequently was observed. This protein having a molecular weight of 34,500 also was purified to homogeneity and appeared to be a proteolytic fragment derived from the regulatory subunit. Limited proteolysis with trypsin converted the regulatory subunit into a protein having a molecular weight of 34,500 and a polypeptide fragment having a molecular weight of approximately 11,000. Although the 34,500 molecular weight protein retained its capacity to bind cyclic AMP, it was monomeric apparently having lost its ability to aggregate to a dimer.     

Unspecific arginine kinase of molecular weight 150 000. Amino acid composition, subunit structure and number of substrate binding sites.

The amino acid composition of unspecific arginine kinase of molecular weight 150 000 of Sabella pavonina muscle has been determined. If was found to be very similar to that of the phosphagen kinases previously studied. The subunit structure of the enzyme has been investigated by physical and chemical means. The data obtained from ultracentrifugation studies in 6 M guanidine hydrochloride and from molecular sieving and disc electrophoresis in 8 M urea, as well as the tryptic peptide mapping, suggest that Sabella muscle kinase is composed of four non-covalently linked polypeptide chains, with similar molecular weights. The number of binding sites for the nucleotide substrate ADP-Mg2+ has been estimated, using differential spectrophotometry and gel filtration on Sephadex columns. By both methods it was demonstrated that the enzyme contains two catalytic sites per protein molecule of molecular weight 150 000. Thus, arginine kinase from Sabella muscle, of molecular weight 150 000, consists of four similar polypeptide chains, but possesses only two substrate binding sites per tetrameric molecule.     

Purification and properties of a phosphorylase (phosphoprotein) phosphatase associated with an alkaline phosphatase of Mr 35000 from bovine adrenal cortex.

A metal-ion-independent, nonspecific phosphoprotein phosphatase (Mr = 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ greater than Mn2+ greater than Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ greater than Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.     

Enhancement of the glutaminase activity of carbamyl phosphate synthetase by alterations in the interaction between the heavy and light subunits.

Glutamine-dependent carbamyl phosphate synthetase (from Escherichia coli) was previously shown to be composed of a light subunit (molecular weight similar to 42,000) which has the binding site for glutamine and a heavy subunit (molecular weight similar to 130,000) which has binding sites for the other reactants and allosteric effectors. The subunits may be separated with retention of catalytic activities; only the separated light subunit exhibits glutaminase activity. The previous finding that storage of the native enzyme at pH 9 at 0 degrees increased its glutaminase activity by about 25-fold was further investigated; such storage markedly decreased the glutamine- and ammonia-dependent synthetase activities of the enzyme. Treatment of the enzyme with p-hydroxymercuribenzoate led to transient increase of glutaminase activity followed by inhibition. When the enzyme was treated with N-ethylmaleimide or with 5,5'-dithiobis-(2-nitrobenzoate), the glutaminase activity was increased by about 250-fold with concomitant loss of synthetase activities. The enhancement of glutaminase produced by storage of the enzyme at pH 9 was associated with intermolecular disulfide bond formation and aggregation of the enzyme. Aggregation also was observed after extensive treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) or N-ethylmaleimide. However, a moderate increase of glutaminase activity (about 30-fold) was observed without aggregation under conditions in which one sulfhydryl group on the light subunit reacted with either reagent. The findings suggest that the increased glutaminase activities observed here are associated with structural changes in the enzyme in which the intersubunit relationship is altered so as to uncouple the catalytic functions of the enzyme and to facilitate access of water to the glutamine binding site on the light subunit.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide