Studies on the subunits of human myeloperoxidase

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.

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Separation and partial characterization of guinea-pig caseins

Biochemical Journal ◽

10.1042/bj1730633 ◽

1978 ◽

Vol 173 (2) ◽

pp. 633-641 ◽

Cited By ~ 14

Author(s):

R K Craig ◽

D McIlreavy ◽

R L Hall

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Guinea Pig ◽

Amino Acid Composition ◽

Acid Composition ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

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Kinetic and physical properties of the l-malate-NAD+ oxidoreductase from Methanospirillum hungatii and comparison with the enzyme from other sources

Biochemical Journal ◽

10.1042/bj1930235 ◽

1981 ◽

Vol 193 (1) ◽

pp. 235-244 ◽

Cited By ~ 9

Author(s):

A C Storer ◽

G D Sprott ◽

W G Martin

Keyword(s):

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Bacterial Species ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Sequential Reaction ◽

Nad Oxidoreductase ◽

Indirect Relationship

The L-malate-NAD+ oxidoreductase of Methanospirillum hungatii was purified to homogeneity by using Blue Sepharose and ADP-Sepharose affinity chromatography. The molecular weight was estimated as 61 700 +/- 1900 by gel filtration and 64 200 +/- 1200 by ultracentrifugation. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein is composed of two polypeptide chains, each corresponding to 31 350 +/- 2150 daltons. Inhibition patterns obtained for malate, alpha-oxoglutarate and ADP established that the sequential reaction mechanism was ordered, with NADH serving as the first substrate. Intracellular concentrations of oxaloacetate approximated the Km value of 27 microM, but NADH was present at less than Km values. Comparison of the amino-acid composition of the L-malate-NAD+ oxidoreductase of M. hungatii and 22 others from prokaryotic and eukaryotic cells revealed a significant direct relationship between average hydrophobicity and the frequency of non-polar side chains, as well as a significant indirect relationship between average hydrophobicity and the polarity ratio. Calculations based on amino-acid-composition data indicated significant composition similarity between pairs of mammalian-cytoplasmic or pairs of mitochondrial L-malate-NAD+ oxidoreductases from various sources, but no significant composition similarity between any of the pairs of bacterial species examined.

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Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma

Biochemical Journal ◽

10.1042/bj2410685 ◽

1987 ◽

Vol 241 (3) ◽

pp. 685-692 ◽

Cited By ~ 129

Author(s):

P Manjunath ◽

M R Sairam

Keyword(s):

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Seminal Plasma ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Terminal Residue ◽

Acidic Proteins

Three major acidic proteins of bovine seminal plasma, BSP-A1, BSP-A2 and BSP-A3, were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. The proteins were purified on the basis of their stimulatory effect on the basal release of gonadotropins by rat anterior-pituitary cells in culture. All three proteins migrated as distinct single bands in the presence or absence of 2-mercaptoethanol in SDS/polyacrylamide-gel electrophoresis. Their Mr values were estimated to be between 15,000 and 16,500 by SDS/polyacrylamide-gel electrophoresis. Similar Mr estimates were obtained when they were subjected to gel filtration on a calibrated column of Sephadex G-75 equilibrated in 0.05 M-acetic acid, pH 3.0. However, BSP-A1 and BSP-A2 were eluted as aggregated molecules (Mr 60,000-120,000) during gel filtration on Sephadex G-200 equilibrated in 0.05 M-NH4HCO3, pH 8.5, or phosphate buffer, pH 7.0, containing 0.15 M-NaCl. In the presence of 8 M-urea both BSP-A1 and BSP-A2 were eluted at positions corresponding to Mr values of 17,000-20,000. BSP-A1 and BSP-A2 had an identical amino acid composition, which differed largely from that of BSP-A3. All three proteins contained aspartic acid as the N-terminal residue, and cysteine was identified as the C-terminal residue. BSP-A1 and BSP-A2 are glycoproteins containing galactosamine, sialic acid and neutral sugars, but BSP-A3 did not contain any covalently attached sugars. Whereas BSP-A2 and BSP-A3 were eluted unadsorbed, BSP-A1 bound to wheat-germ lectin-Sepharose 6MB and could be eluted by the competing sugar N-acetyl-D-glucosamine. Treatment of BSP-A1 and BSP-A2 with trypsin resulted in complete loss of gonadotropin-release activity, but BSP-A3 retained full activity. Antibody raised against BSP-A1 did not cross-react with BSP-A3, or vice versa. All these properties indicated marked structural differences between BSP-A3 and BSP-A1 (or BSP-A2). On the basis of amino acid composition it was concluded that BSP-A1, BSP-A2 and BSP-A3 are the same as the gonadostatins [Esch, Ling, Bohlen, Ying & Guillemin (1983) Biochem. Biophys. Res. Commun. 113, 861-867].

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Isolation of the smallest component of silk protein

Biochemical Journal ◽

10.1042/bj1870413 ◽

1980 ◽

Vol 187 (2) ◽

pp. 413-417 ◽

Cited By ~ 29

Author(s):

S Tokutake

Keyword(s):

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Silk Protein ◽

Silk Proteins ◽

Cupric Hydroxide

Silk proteins were solubilized from cocoons with ethylenediamine/cupric hydroxide solution. A series of polymers of the smallest component, detected by polyacrylamide-gel electrophoresis, could be converted into the smallest component by reduction and aminoethylation. Fibroin and sericin fractions were separated by precipitation of sericin at pH 5.5. On gel electrophoresis, sericin showed distinct bands but fibroin did not. The components of fibroin and sericin were fractionated by gel filtration on Sepharose 6B. The smallest component in the sericin fraction was purified by rechromatography and showed a single band on gel electrophoresis. Its mol. wt. was 24 000, and its amino acid composition was determined.

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Isolation, preparation and the amino acid composition of 4 milk-fat globule membrane proteins solubilized by treatment with sodium dodecyl sulphate

Journal of Dairy Research ◽

10.1017/s0022029900015983 ◽

1976 ◽

Vol 43 (3) ◽

pp. 401-409 ◽

Cited By ~ 11

Author(s):

T. E. Cawston ◽

M. Anderson ◽

G. C. Cheeseman

Keyword(s):

Amino Acid ◽

Membrane Proteins ◽

Amino Acid Composition ◽

Acid Composition ◽

Milk Fat ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Milk Fat Globule Membrane ◽

Milk Fat Globule ◽

Fat Globule

SummaryMilk-fat globule membrane (MFGM) proteins were solubilized by treatment with SDS. Four of the major proteins were isolated as SDS complexes using column chromatography. The purity of each isolate was determined by SDS polyacrylamide gel electrophoresis and sufficient of each protein was obtained for amino acid analysis. The amino acid compositions of the isolated MFGM proteins and a total MFGM protein extract were determined. Differences in amino acid composition were found in particular between the major MFGM glycoprotein and the other 3 membrane proteins. The relationships of the amino acid composition to protein properties and structure are discussed.

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Characterization of amylases produced by tumors.

Clinical Chemistry ◽

10.1093/clinchem/27.4.556 ◽

1981 ◽

Vol 27 (4) ◽

pp. 556-559 ◽

Cited By ~ 15

Author(s):

T Takeuchi ◽

H Fujiki ◽

T Kameya

Keyword(s):

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Tertiary Structure ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Relative Molecular Mass ◽

Major Peak ◽

Salivary Amylase ◽

A Minor

Abstract Amylases were purified and characterized from three amylase-producing human tumors. The relative molecular mass of the amylases was estimated to be 54 000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, different from human salivary amylase (61 000 and 64 000) and human pancreatic amylase (60 000). The tumor amylases had completely identical antigenicities with human salivary and pancreatic amylases against antibody to human salivary amylase, while the intensity of the tumor amylases was less than 20% of that of human salivary and pancreatic amylases on single radial immunodiffusion. On isoelectric focusing, two of the three tumor amylases showed a major peak at pH 6.4, which corresponded to a major peak of human salivary amylase, the other showed a major peak at pH 6.4, which corresponded to a minor peak of human salivary amylase. The three tumor amylases showed similar amino acid composition, different from those of human salivary and pancreatic amylases. These findings suggest that tumor amylases have a tertiary structure similar to that of normal human amylases, but differ from them in amino acid composition.

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2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation byRhodopseudomonas palustris

Journal of Bacteriology ◽

10.1128/jb.180.9.2330-2336.1998 ◽

1998 ◽

Vol 180 (9) ◽

pp. 2330-2336 ◽

Cited By ~ 40

Author(s):

Dale A. Pelletier ◽

Caroline S. Harwood

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Mass ◽

Coenzyme A ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Ring Cleavage ◽

Benzoate Degradation ◽

Coa Hydrolase

ABSTRACT 2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 μmol min−1 mg of protein−1. Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.

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Comparison between the monomeric and binary-complex forms of procarboxypeptidase A from whole pig pancreas

Biochemical Journal ◽

10.1042/bj1970141 ◽

1981 ◽

Vol 197 (1) ◽

pp. 141-147 ◽

Cited By ~ 18

Author(s):

M C Martínez ◽

F X Avilés ◽

B Sansegundo ◽

C M Cuchillo

Keyword(s):

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Enzymic Activity ◽

Binary Complex ◽

Activation Process ◽

Carboxypeptidase A ◽

Iodosobenzoic Acid

Two different forms of procarboxypeptidase A (I and II) were obtained from pig pancreas extracts. The Mr values, the pattern found on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, and the sedimentation coefficients indicate that form I is a binary complex formed by two different subunits, whereas form II is a monomer. The carboxypeptidase A-precursor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx. 12500) and, in the case of the binary complex, the activation with trypsin follows a rather complex pattern, suggesting that the accompanying subunit of form I might play a modulating role in the activation process. Although the appearance of enzymic activity is rather slow, a protein with an Mr equivalent to that of active carboxypeptidase A is found very early in the activation process. Both zymogens are glycoproteins (so far no carbohydrate has been reported in any procarboxypeptidase A) and both contain two strongly bound Zn2+ ions/molecule. Other chemical and physical properties were also determined.

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Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

Biochemical Journal ◽

10.1042/bj1970629 ◽

1981 ◽

Vol 197 (3) ◽

pp. 629-636 ◽

Cited By ~ 19

Author(s):

J L McKenzie ◽

A K Allen ◽

J W Fabre

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Human Brain ◽

Affinity Chromatography ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Antibody Affinity ◽

Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

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A metalloproteinase extracellularly released byCrithidia deanei

Canadian Journal of Microbiology ◽

10.1139/w03-081 ◽

2003 ◽

Vol 49 (10) ◽

pp. 625-632 ◽

Cited By ~ 15

Author(s):

Claudia Masini d'Avila-Levy ◽

Rodrigo F Souza ◽

Rosana C Gomes ◽

Alane B Vermelho ◽

Marta H Branquinha

Keyword(s):

Molecular Mass ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Residual Activity ◽

Major Protein ◽

Motile Cells ◽

Sds Page ◽

Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The incorporation of gelatin into SDSPAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

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