Folylpolyglutamate synthetase from beef liver: purification and some properties.

The polypeptide chain of folylpolyglutamate synthetase from beef liver has been isolated and partially characterized. This polypeptide has an apparent molecular weight of 73,000. Its amino-terminal residue is blocked. Amino acid analysis agrees with the hydrophobic properties and the pI (6.0) of this cytosolic enzyme. Polyclonal antibodies to the denatured enzyme have been prepared.     

Studies on cytochrome c oxidase, V. Polypeptide IV: alignment and amino acid sequences on cyanogen bromide fragments.

The isolation and chemical characterization of polypeptide IV from beef heart cytochrome oxidase is described. The protein is one of the main (stoichiometric) components of the oxidase. It is the largest polypeptide of the enzyme synthezised in the cytoplasm and has, as such, also been identified in enzyme preparations from yeast and Neurospora. A partial sequence, consisting of 105 amino acid residues which give a frame work of the covalent structure of the polypeptide is obtained from N- anc C-terminal sequencing and from the cyanogen bromide fragments of the chain. The isolation and sequencing of the fragments of this membrane protein are discussed.     

Effect of amino acid substitutions in the polypeptide chain of DNA-polymerase on manifestation of the mutator effect]

Thin map of gene 43, controlling the synthesis of T4 DNA polymerase, is obtained by mapping experiments performed with 39 amber mutants, and is used for analysis of the sites of DNA polymerase gene from the point of view of displaying the mutator effect. The mutant sites studied possessed different reaction on amino acid substitutions in the polypeptide chain of the enzyme. Most of sites of the DNA polymerase gene, with the exception of two "supersensitive", responsed only on the apparent type of the amino acid substitutions: the mutator effect of amber mutations, which are located at these sites, was exhibited only in the case of insertion of the definite amino acid in the respective point of polypeptide chain. The proposed system of amber mutations for studying the mutator effect, allowed the authors to obtain the data on the effect of concrete alterations in the polypeptide chain of the enzyme on the development of its mutator properties.     

Rabbit muscle phosphoglucomutase is a monomer

A procedure which avoids the use of heat or precipitation with salt at acid pH has been developed for the isolation of phosphoglucomutase from rabbit muscle. Contrary to earlier reports, the enzyme obtained by this procedure as well as by published techniques is a single polypeptide chain. This is true of phosphorylated as well as dephosphorylated proteins.     

Studies of glutamate dehydrogenase: analysis of functional areas and functional groups.

1. It is shown by limited tryptic digestion of beef liver glutamate dehydrogenase under native conditions that the amino terminus of the polypeptide chain is located at the surface of the molecule. End-group analysis after trypsin treatment yields aspartic acid as the new N-terminal amino acid while the C-terminal threonine remains unchanged. 2. NADH, especially in the presence of 2-oxoglutarate, protects the enzyme against tryptic degradation. In the absence of the coenzyme, glutamate dehydrogenase is rapidly inactivated. 3. The regulatory effects of ADP and GTP are only slightly altered by trypsin. A small shift of the pH dependence of the activation by ADP is observed. 4. The quaternary structure of the unimer of the enzyme is not affected by limited tryptic digestion indicating that the N-terminal part of the polypeptide chain is not located in the contact domains between the polypeptide chains. The association of the hexamer to large associated particles is reduced but not abolished. 5. It is shown by treatment of the enzyme with iodo[2(-14)C]acetic acid as well as with Ellman's reagent that the six - SH groups of the polypeptide chain are buried and not accessible to these reagents in phosphate buffer. In Tris buffer they become exposed and react in the order 89, 55, 197, 115, 270, 319. This together with the result that in Tris buffer the rat of inactivation caused by trypsin is higher than in phosphate buffer indicates that Tris buffer changes drastically the properties of the enzyme. 6. Cross-linking of the enzyme molecule with bifunctional reagents and subsequent dodecylsulfate-polyacrylamide electrophoresis shows that the six identical polypeptide chains are arranged in two groups of three. 7. The implications of these results for the tertiary and quaternary structure of beef liver glutamate dehydrogenase are discussed.     

The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa.

The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.     

Glucose-6-phosphate dehydrogenase. Characteristics revealed by the rat liver enzyme structure

The primary structure of glucose-6-phosphate dehydrogenase from rat liver has been determined, showing the mature polypeptide to consist of 513 amino acid residues, with an acyl-blocked N-terminus. This structure is homologous to those of both other eutherian and marsupial mammals (human and opossum), thus characterizing a mammalian type enzyme to which the human form, notwithstanding its large number of genetic variants, conforms. The mammalian type differs from the fruit fly enzyme by about 50%. Known mutant forms exhibit further differences, widely distributed along the polypeptide chain. Structural patterns show glucose-6-phosphate dehydrogenases to consist of a few variable regions intermixed with relatively constant segments.     

The covalent structure of hepatic microsomal epoxide hydrolase. II. The complete amino acid sequence

The complete amino acid sequence of hepatic microsomal epoxide hydrolase has been determined. The protein contains 455 amino acid residues in a single polypeptide chain and has Mr = 52,691. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. This is the first epoxide hydrolase and the third microsomal enzyme for which the complete sequence has been determined.     

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.     

Physicochemical properties of a fibrinolytic enzyme from Streptomyces thermovulgaris culture broth

Some properties of fibrinolytic enzyme from cultural fluid of Streptomyces thermovulgaris have been studied. The molecule of enzyme has been shown to consist of one polypeptide chain with molecular mass 28000 dalton, pi = 7.45-7.6. The amino acid composition of protein is determined, the protein does not contain cysteine residues. The enzyme is not thermostable, and Ca2+ ion does not exert stabilized influence. In opposition to diisopropylfluorophosphate, phenylmethylsulfonyl fluoride does not inhibit the enzyme activity.     

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family.     

Chromophore activating enzyme involved in the biosynthesis of the mikamycin B antibiotic etamycin from Streptomyces griseoviridus

A 3-hydroxypicolinic acid activating enzyme from etamycin producing Streptomyces griseoviridus has been purified to apparent homogeneity. Etamycin is a member of mikamycin B antibiotics, chromopeptide lactones, which contain 3-hydroxypicolinic acid (3-HPA) as the chromophoric group. The enzyme catalyzes both the 3-HPA-dependent ATP-pyrophosphate exchange and the formation of 3-HPA adenylate from 3-HPA and ATP. SDS-polyacrylamide gel electrophoresis indicates that the enzyme is a single polypeptide chain with a Mr between 56,000 and 58,000. The molecular mass of the native enzyme was in the same range. In addition to 3-HPA, the enzyme catalyzes the formation of adenylates from picolinic acid, nicotinic acid, and 2-pyrazinecarboxylic acid. Nicotinic acid and picolinic acid when added externally to etamycin producing S. griseoviridus cultures gave rise to the formation of etamycin analogues each containing nicotinic acid or picolinic acid instead of the genuine 3-HPA. The data strongly suggest that the enzyme is involved in the biosynthesis of the chromopeptide lactone etamycin and possibly in that of other mikamycin B antibiotics.     

The distribution of carbohydrate side chains along the polypeptide chain of glucoamylase

Glucoamylase is a starch-hydrolyzing enzyme with a glycoprotein structure, used industrially for the conversion of starch to glucose, citric acid, corn syrups, and high-fructose sweeteners. This enzyme possesses an unusual type of structure in which many carbohydrate side chains are linked O-glycosidically to serine and threonine residues of the polypeptide chain. The carbohydrate side chains may be single monosaccharide residues or oligosaccharides of mannose, glucose, galactose, and in some cases N-acetylglucosamine. New data from experiments on the CNBr fragmentation of glucoamylase followed by chemical and immunological characterization of the fragments show that the carbohydrate side chains are distributed randomly along the polypeptide chain. Such a structure is appropriately termed a random model reprensentation for the glucoamylase molecule.     

Structural characteristics of prostaglandin synthetase from sheep vesicular gland

Prostaglandin synthetase contains both oxygenase and peroxidase activity and catalyzes the first step of prostaglandin synthesis. Aspirin (acetylsalicylic acid) inhibits oxygenase activity by acetylating a serine residue of the enzyme. In the current study, we have investigated the subunit structure of this complex enzyme and the stoichiometry of aspirin-mediated acetylation of the enzyme. The enzyme was purified to near homogeneity in both active and aspirin-acetylated forms. The purified protein was analyzed for enzymatic activity, [3H]acetate content following treatment with [acetyl-3H]aspirin, NH2-terminal sequence, and amino acid composition. The results show first, that the enzyme can be purified to near homogeneity in an active form; second, that the enzyme consists of a single polypeptide chain (molecular weight 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a unique NH2-terminal sequence (Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-Met-Gly-); and third, that aspirin inhibits the enzyme by transfer of one acetate per enzyme monomer. Therefore, the two distinct enzymatic activities, oxygenation and peroxidation, are present in a single polypeptide chain. Experiments with a cross-linking agent indicate that in nonionic detergent the enzyme is a dimer of two identical subunits.     

Acetylation of prostaglandin endoperoxide synthetase with acetylsalicylic acid

Incubation of purified prostaglandin endoperoxide synthetase from sheep vesicular glands with aspirin results in a covalent binding of the acetyl group of acetylsalicylic acid to the protein. During this acetylation, the cyclooxygenase activity is lost, but not the peroxidase activity. The reaction is completed when almost one acetyl group is bound per polypeptide chain (Mr = 68 000). After proteolysis of [3H]acetyl-protein with pronase, radioactive N-acetylserine was obtained. Originally, however, the hydroxyl group of an internal serine residue in the chain is acetylated. The formation of N-acetylserine can be explained by a rapid O leads to N acetyl shift as soon as the NH2 group of serine is liberated. A radioactive dipeptide was isolated from a thermolysin digest of the [3H]acetyl-enzyme containing phenylalanine and serine, phenylalanine being its N-terminal amino acid. Automatic Edman degradation of native and acetylated enzyme showed that only one polypeptide sequence was present: Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-X-X-Tyr-. The N-terminal sequence has an apolar character.     

Purification and Properties of a Highly Active Organophosphorus Acid Anhydrolase from Alteromonas undina

A highly active organophosphorus acid anhydrolase from Alteromonas undina was purified to homogeneity and found to be composed of a single polypeptide chain with a molecular weight of 53,000. With diisopropylfluorophosphate as a substrate, the purified enzyme has a specific activity of ~575 μmol/min/mg of protein. The enzyme has optimum activity at pH 8.0 and 55°C and is stimulated by sulfhydryl reducing agents and manganese. It is capable of rapidly hydrolyzing a wide range of nerve agents and several chromogenic phosphinates.     

Rat muscle 5'-adenylic acid aminohydrolase. I. Purification and subunit structure.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) has been purified to apparent homogeneity from rat muscle. The preparation exhibits a single polypeptide band with a molecular weight of 60,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a sedimentation coefficient of 11.3 S. Analysis by sedimentation equilibrium techniques showed the nat-ive enzyme to have a molecular weight of 238,000, whereas the enzyme, when analyzed in 6 M guanidine hydrochloride and 10 mM 2-mercaptoethanol, had a molecular weight of only 59,500. The amino acid composition of the enzyme was determined and peptide mapping was performed on a tryptic digest of S-carboxymethylated enzyme. NH2-terminal analysis by both the dansylation and cyanate procedures failed to identify a free NH2 terminus. Treatment of the enzyme with carboxypeptidase A resulted in the release of approximately 0.5 mol each of valine and leucine per 60,000 g of enzyme. The data presented indicate that hte native enzyme has a tetrameric structure consisting of four polypeptide chains each having a molecular weight of 60,000. The COOH-terminal analysis can be interpreted either as an indication of subunit heterogeneity or as a result of incomplete digestion of a -X-Leu-Val sequence at the end of a single type of polypeptide chain. Tryptic peptide maps strongly support the latter interpretation and suggest that the subunits are essentially identical.     

Complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp

The complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp. has been determined by analysis of peptides after cleavage with lysyl endopeptidase, pepsin and chymotrypsin. The protein consists of a single polypeptide chain consisting of 267 amino acid residues and a molecular mass of 27972 Da. The sequence of Flavobacterium endo-beta-N-acetylglucosaminidase is very close to that of the Streptomyces enzyme (endo-H), having 60% similarity and very similar hydropathy profiles. Similarities were also found between Flavobacterium endo-beta-N-acetylglucosaminidase and chitinases from Bacillus circulans, Serratia marcescens and Phaseolus vulgaris.     

Amino acid sequence of guinea pig prostate kallikrein

The primary structure of the major arginine esteropeptidase from guinea pig prostate has been deduced from automated Edman degradation of peptides generated by clostripain, cyanogen bromide, endoproteinase Lys-C, and Staphylococcus aureus V8 protease digestion of the protein. The esteropeptidase is a single polypeptide chain comprised of 239 amino acids and contains 2 apparent sites of carbohydrate attachment, Asn-78 and Asn-169. Both occur in consensus sequences for N-linked glycosylation sites. The esteropeptidase exhibits approximately 35% homology with trypsin including conservation of the catalytic residues and the aspartic acid which confers specificity toward basic amino acids. The sequence identity, however, extends to greater than 60% with the kallikrein family of serine proteases. In addition to the overall homology, the guinea pig enzyme displays a number of features characteristic of kallikreins including 10 conserved half-cystine residues, a C-terminal proline, and the "kallikrein loop". On the basis of this structural relatedness, the enzyme has been designed as guinea pig prostate kallikrein. In contrast to many of the kallikreins of other species and tissues, this enzyme does not contain any sites within the kallikrein loop sensitive to proteases that result in internal breaks in the polypeptide chain.