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

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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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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Studies on the subunits of human myeloperoxidase

Biochemical Journal ◽

10.1042/bj2220701 ◽

1984 ◽

Vol 222 (3) ◽

pp. 701-709 ◽

Cited By ~ 52

Author(s):

R L Olsen ◽

C Little

Keyword(s):

Heat Treatment ◽

Amino Acid ◽

Molecular Mass ◽

Amino Acid Composition ◽

Acid Composition ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Additional Species ◽

Main Protein

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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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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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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Isolation of Labeled Lipoprotein from Escherichia coli and Proteus mirabilis after Incubation with [14C]Penicillin

Zeitschrift für Naturforschung C ◽

10.1515/znc-1985-5-622 ◽

1985 ◽

Vol 40 (5-6) ◽

pp. 415-420 ◽

Cited By ~ 1

Author(s):

Gerhard Gruner ◽

Monier H. Tadros ◽

Roland Plapp

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Proteus Mirabilis ◽

Polyacrylamide Gel Electrophoresis ◽

Apparent Molecular Weight ◽

Penicillin G ◽

Free Form ◽

E Coli

Abstract [14C]penicillin binding experiments and membrane analysis were carried out with cell envelope preparations from Escherichia coli and Proteus mirabilis. After incubation with [14C] penicillin G labeled free lipoprotein could be identified. The analysis of the isolated lipoprotein by SDS polyacrylamide gel electrophoresis indicates that there is only one protein with an apparent molecular weight of 7000. The amino acid composition of isolated labeled free lipoprotein from E. coli was identical to the lipoprotein already found in E. coli. It is a point of interest that the amino acid composition of the isolated labeled free lipoprotein from P. mirabilis D52 differs from that found in other mutants of this strain. The free form of lipoprotein from P. mirabilis D52 is composed of 61 amino acids and has glycine, phenylalanine and proline as specific components.

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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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Changes in renal glomerular basement membrane with age and nephritis

Canadian Journal of Biochemistry ◽

10.1139/o77-179 ◽

1977 ◽

Vol 55 (12) ◽

pp. 1197-1206 ◽

Cited By ~ 15

Author(s):

N. Kalant ◽

S. Satomi ◽

R. White ◽

E. Tel

Keyword(s):

Amino Acid ◽

Basement Membrane ◽

Amino Acid Composition ◽

Acid Composition ◽

Glomerular Basement Membrane ◽

Disulfide Bonds ◽

Sodium Dodecyl ◽

Collagen Content ◽

Insoluble Residue ◽

Adult Rats

Glomerular basement membrane was obtained from normal, young and old adult rats and from animals with antiserum nephritis, daunomycin nephrosis, and lathyrism. With increased age there was an increase in the collagen content of whole glomeruli and of the basement membrane. About 50% of the membrane protein was solubilized in 1% sodium dodecyl sulfate, and a further 35% was solubilized by reduction and alkylation. Inhibition of formation of collagen cross-links by induction of lathyrism did not affect membrane solubility. Preparative disc gel electrophoresis permitted separation of a number of components of different composition; with decreasing molecular weight the collagen content declined from almost 100% to 0%.In antiserum nephritis, there was an increase in the noncollagen components of the membrane and marked alterations in amino acid composition, both of whole membrane and of electrophoretically separated components. In daunomycin nephrosis, the amino acid composition was similar to that of antiserum nephritis. The solubility of membrane from nephritic rats was normal. The composition of the insoluble residue was similar for all membrane preparations and resembled pure collagen. It is suggested that the presence of abnormal noncollagen proteins, associated with the insoluble collagen core by hydrophobic and disulfide bonds, as in antiserum nephritis, is associated with increased membrane permeability leading to proteinuria.

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Purification and properties of a proteolytic enzyme from the cercariae of the human trematode parasite Schistosoma mansoni

Biochemical Journal ◽

10.1042/bj2010137 ◽

1982 ◽

Vol 201 (1) ◽

pp. 137-144 ◽

Cited By ~ 31

Author(s):

W J Landsperger ◽

M A Stirewalt ◽

M H Dresden

Keyword(s):

Amino Acid ◽

Schistosoma Mansoni ◽

Amino Acid Composition ◽

Acid Composition ◽

Isoelectric Point ◽

Proteolytic Enzymes ◽

Sodium Dodecyl ◽

Skin Penetration ◽

Cathepsin G ◽

Trematode Parasite

Skin penetration by the cercarial stage of the human trematode parasite Schistosoma mansoni is mediated by the secretion of proteolytic enzymes able to digest components of mammalian connective tissues. In the present study the purification of these proteinases from cercarial homogenates is reported. The major proteinase species has a mol. wt. of approx. 25 000 and exists in monomeric form as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This proteinase has an isoelectric point of 6.0. Studies presented here, with a variety of substrates and inhibitors, confirm previous claims that these proteinases belong to the serine class, and, in addition, suggest that they resemble the vertebrate chymotrypsins rather than trypsins or elastases. However, the amino acid composition of the cercarial proteinase differs significantly from bovine chymotrypsin and from the human leucocyte chymotrypsin-like cathepsin G. The amino-acid-composition differences between these proteinases are consistent with their differences in isoelectric point. In order to obtain an insight into the role of the proteinase in skin penetration, its activity on cartilage proteoglycan monomers and on the isolated peptide backbone of proteoglycan was studied. The results of the present study indicate that the cercarial enzyme catalyses a limited specific digestion of the peptide core.

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