alpha-Amylase inhibitor from fungus Cladosporium herbarum F-828

A strain of fungus Cladosporium herbarum extracellularly produced an inhibitor specific for mammalian alpha-amylase. The inhibitor was purified 81-fold by freeze-thawing, heat treatment, and column chromatography on DEAE-cellulose, Sephadex G-75, DEAE-Sephacel, and Bio-Gel P-100. An apparent molecular weight of approximately 18,000 was estimated for the inhibitor using Bio-Gel P-100 filtration. The purified inhibitor preparation was a glycoprotein containing about 10% carbohydrate. The amino acid analysis of the inhibitor showed abundances of Gly, Asp, Glu, Ser, Ala, and Thr residues. The inhibitor was stable between pH 5 and 12 at 4 degrees C, and below 80 degrees C at pH 7.0. A binary complex formation out of equimolar amounts of the inhibitor and alpha-amylase, was demonstrated by polyacrylamide gel electrophoresis, and Bio-Gel P-100 chromatography. Kinetic studies exhibited that the inhibitor noncompetitively inhibited the enzyme reaction with a Ki value of 2.3 approximately 4.8 x 10(-10) M, by combining with the enzyme molecule at a different site from the substrate binding site.     

The proteolytic system of Penicillium roqueforti. V. -- Purification and properties of an alkaline aminopeptidase.

An alkaline aminopeptidase was isolated from the culture medium of Penicillium roqueforti. The enzyme was purified by ammonium sulfate precipitation, filtration on Bio-Gel P-100, chromatography on D.E.A.E.-cellulose and hydroxylapatite, filtration on Bio-Gel P-150 and electrofusing. The purified preparation was homogeneous on polyacrylamide gel electrophoresis at pH 8.5. The molecular weight of the enzyme was estimated to be about 35,000 daltons. The isoelectric point is 4.5. The optimum pH for L-leucine-p-nitroanilide hydrolysis is 8.0. At 35 degrees C the enzyme is stable between pH 6.0 and 7.0. Ethylenediamine tetraacetic acid and a sulfhydryl reagent (p-hydroxymercuribenzoate) inhibit the activity, but the enzyme is insensitive to diisopropylfluorophosphate. Hydrolysis of synthetic peptides shows that the enzyme releases apolar amino acids. Dipeptides are poorly hydrolyzed and Gly in penultimate or N-terminal position causes poor activity. The enzyme is able to cleave the N-terminal Arg-Pro bond of bradykinin.     

Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

Excretion of a protease by Serratia marcescens

Excretion of an extracellular protease of Serratia marcescens ATCC 25419 occurred during logarithmic growth and was highest (per cell) when cultures reached the stationary growth phase. Production of the extracellular protease was induced by leucine or casein in minimal medium or by growth in tryptone-yeast medium. In the late stationary phase an intracellular protease activity accumulated which was also observed in mutants with very low extracellular protease activity. The excreted protease was the dominant protein in the growth medium. The protease was purified to homogeneity by column chromatography on Bio-Gel P-100 and on DEAE-cellulose. Quantitative amino acid analysis revealed the absence of sulfurcontaining amino acids. The enzyme consists of one polypeptide chain. A molecular weight of 51,000 and 55,000 was estimated using polyacrylamide gel electrophoresis and chromatography on Bio-Gel P-100 respectively. The enzyme cleaved only N--benzoyl-DL-lysine-and-arginine-nitroanilides but not the corresponding leucine or tyrosine derivatives nor a set of diand tripeptides.Abbreviation SDS sodium dodecylsulfate     

Isolation of an N-acetyl-d-galactosamine-binding protein from winged bean (Psophocarpus tetragonolobus)

A lectin has been purified to homogeneity by affinity chromatography on a Sepharose-N-caproyl-d-galactosamine column from the local variety of winged bean (Psophocarpus tetragonolobus). The lectin agglutinated native erythrocytes of all blood groups. This hemagglutination was inhibited best by N-acetyl-d-galactosamine. A molecular weight of 41,000 was obtained for the lectin by gel filtration on Bio-Gel P-100. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis of the same lectin showed a single M_r 35,000 polypeptide.     

Chitin Coated Cellulose as an Adsorbent of Lysozyme-like Enzymes: Some Applications

Galactose oxidase was purified from the culture supernatant of Gibberella fujikuroi by ammonium sulfate precipitation, chromatographies on DEAE-cellulose and hydroxylapatite, and gel filtration on Bio-Gel P-100. The purified enzyme had a molecular weight of 90,000 and an isoelectric point of pH 3.7, and contained about one atom of copper and about one atom of iron per mol of the enzyme protein. The enzyme was markedly inactivated by a copper-chelating agent, diethyldithiocarbamate, and reducing agents. The apoenzyme preparing on treatment of the enzyme with diethyldithiocarbamate could be reactivated only by the addition of either Cu⁺ or Cu^{2 +}. These results indicate that copper is involved in galactose oxidase activity of G. fujikuroi.     

High-molecular-weight cadmium-binding proteins in rat liver cytosol: isolation and partial characterization of an approximately 50-kDa protein

The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.     

Purification of interferon from mouse Ehrlich ascites tumor cells

Interferon production was induced in mouse Ehrlich ascites tumor cells by infection with Newcastle disease virus. The interferon produced was purified by precipitation with ammonium sulfate, chromatography on carboxymethyl-Sephadex, treatment with blue dextran and polyethylene glycol, gel filtration on Bio-Gel P-60 and Bio-Gel P-200, chromatography on phosphocellulose, isoelectric focusing, and chromatography on octyl-Sepharose. The specific activity of the product was 1.6 x 10(9) NIH mouse interferon reference standard units/mg of protein. Electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate indicated that the apparent molecular weight of the interferon-active material ranged from 25,000 to 35,000. As revealed by staining the gels with Coomassie brilliant blue, the interferon activity co-migrated with the major, broad protein band. Minor, stainable bands of proteins were free of interferon activity and their apparent molecular weight was smaller than 12,000.     

Isolation, purification, and partial characterization of type V-A hemagglutinin from Escherichia coli GV-12, O1:H-

A hemagglutinin which specifically agglutinates human type A erythrocytes (mannose resistant) was isolated from the growth medium of cultures of Escherichia coli GV-12, serotype O1:H-, and purified by chromatography on Bio-Gel A-1.5 and DEAE-Sephadex A-25. The purity of the hemagglutinin was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. N-terminus analysis indicated that only asparagine resides on the amino terminus. The native hemagglutinin is an aggregate exhibiting a sedimentation coefficient of 9.25, which corresponds to a molecular weight of approximately 200,000. The monomeric molecular weight was found to be approximately 16,300. Amino acid analysis indicated that the hemagglutinin consists of 131 residues, corresponding to a molecular weight of 13,400.     

Purification of the beta-glucosidase from Sclerotinia sclerotiorum

A beta-glucosidase (EC 3.2.1.21) has been isolated from culture filtrates of the fungus Sclerotinia sclerotiorum. The protein was purified by gel filtration on a column of Bio-Gel P-300 and by ion exchange chromatography on DEAE-Bio-Gel A. The molecular weight, determined by gel filtration, was 240,000. Km values for the enzyme towards p-nitrophenyl-beta-D-glucoside and cellobiose were respectively 0.10 mM and 1.23 mM. The beta-glucosidase activity was found to be strongly associated with a beta-xylosidase (EC 3.2.1.37) activity, suggesting that both activities could be represented in a single protein complex.     

Clovamides; l-Dopa Conjugated with trans- and cis-Caffeic Acids in Red Clover (Trifolium pratense)

Selenium-independent glutathione peroxidase was purified from a cell-free extract of Mucor hiemalis by ammonium sulfate fractionation, column chromatographies on DEAE-Sephadex and hydroxylapatite, and gel filtration on Bio-Gel P-100. The purified enzyme was homogeneous on ultracentrifugation. The enzyme had a molecular weight of 45,000 and an isoelectric point of 5.2. The enzyme could reduce cumene hydroperoxide and t-butyl hydroperoxide, but could not reduce hydrogen peroxide. The enzyme was highly specific for glutathione as a hydrogen donor. Mucor glutathione peroxidase was proved to be different from mammalian selenium-dependent glutathione peroxidase I and selenium-independent glutathione peroxidase II in some physicochemical and enzymatic properties.     

Purification and characterization of a hemagglutinin from Arachis hypogeae.

A. hypogaea hemagglutinin was purified by ammonium sulfate fractionation and Sepharose 6 B column chromatography. The homogeneity of the purified hemagglutinin was ascertained by ultracentrifugal analysis and polyacrylamide gel electrophoresis. It has a molecular weight of 106.500 and is a tetramer of a subunit with a molecular weight of 27.000. The purified hemagglutinin agglutinated neuraminidase-treated human erythrocytes regardless of their ABO group type, but did not agglutinate intact erythrocytes. In hapten inhibition assays with simple sugars, the so-called M?kel?'s group 2 sugars, which bear the same configuration of hydroxy groups at C-3 and C-4 as D-galactopyranose, were inhibitors for this hemagglutinin. It does not contain any carbohydrate, in contrast to most phytohemagglutinins except concanavalin A and wheat germ agglutinin.     

Studies on algal cytochromes VI: some properties and amino acid sequence of cytochrome c6 from a green alga, Bryopsis maxima

A photosynthetic c-type cytochrome, cytochrome c6, was extracted from a green alga, Bryopsis maxima, by cutting and immersing the frozen thalli in phosphate buffer, pH 7.0, and purified by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography and Bio-Gel P-10 gel filtration. The ferrcytochrome c6 has absorption maxima at 553.5 (alpha), 523 (beta), 417 (gamma), 318 (delta), and 275 nm, and the ferricytochrome at 695, 528, and 411 (gamma). The molecular weight was estimated to be about 10,000 from Sephadex G-75 gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The midpoint redox potential for the cytochrome was determined by equilibrium titration with a ferro- and ferricyanide system to be 0.385 volt at pH 7.0. Isoelectric points for ferro- and ferricytochromes were determined by density gradient isoelectric focusing electrophoresis to be at pH 3.91 and 4.02, respectively. The complete amino acid sequence of the cytochrome was determined by Edman degradation and by carboxypeptidase digestions of the Cm-cytochrome, 6 staphylococcal protease peptides and 5 lysyl endopeptidase peptides. The cytochrome contained 88 amino acid residues, giving a molecular weight of 9,904 including 1 mol of heme c. The sequence is as follows: GGDLEIGADVFTGNCAACHAGGANSVEPLKTLNKEDVTKYLDGGLSIEAITSQVRNGKGAMPAWSDRLD DEEIDGVVAYVFKNINEGW. A phylogenetic tree of 13 algal cytochromes c6 was constructed by comparing the amino acid differences.     

Identification and purification of a new staphylococcal enterotoxin, H.

A staphylococcal enterotoxin which elicited an emetic response in monkeys but did not share antigenic determinants with any of the identified enterotoxins was identified and purified from Staphylococcus aureus FRI-569. The emetic activity of this new enterotoxin was neutralized only by antibodies specific to it and not by antibodies to enterotoxins A, B, C, D, and E or toxic shock syndrome toxin 1. Immunodiffusion assays did not detect cross-reactivity between this new and all the other identified enterotoxins. The purification procedure involved removal of the enterotoxin from culture supernatant fluids by batch adsorption with CG-50 resin, CM-Sepharose FL ion-exchange chromatography, and Sephacryl 100 HR and Bio-Gel P-30 gel filtration. The molecular weight of this enterotoxin, 27,300, determined by gel filtration on Sephacryl 100 HR agreed with the molecular weight, 28,500, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent migration of this enterotoxin determined by SDS-PAGE did not shift in the presence of a disulfide reducing agent, indicating that it is composed of a single-chain protein. The N-terminal amino acid sequence of the enterotoxin was determined to be Glu-Asp-Leu-His-Asp-Lys-Ser-Glu-Leu-Thr-Asp-Leu-Ala-Leu-Ala-Asn-Ala-Tyr- Gly- Gln-Tyr-Asn-His-Pro-Phe-Ile-Lys-Glu-Asn-Ile, which did not match the N-terminal sequences of any known proteins. The isoelectric point of the enterotoxin determined by isoelectric focusing was about 5.7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of an exoglucanase from Streptomyces flavogriseus

Streptomyces flavogriseus, a mesophilic actinomycete, produces high levels of extracellular enzymes capable of hydrolyzing cellulose and xylan. One such enzyme, an exoglucanase, has been purified to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel permeation chromatography on Bio-Gel P-60, preparative isoelectric focusing, and concanavalin A affinity chromatography. This purification sequence disclosed the presence of several distinct endoglucanase and xylanase fractions. Homogeneity of the purified enzyme was demonstrated by analytical isoelectric focusing and sodium dodecyl sulphate--polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 45 000 and an isoelectric point of 4.15. The enzyme demonstrated negligible activity with carboxymethylcellulose as the substrate. It was able to extensively hydrolyse acid-swollen cellulose; the main product of enzyme action was cellobiose.     

Rainbow trout protamines. Amino acid sequences of six distinct proteins from a single testis

All the protamines present in detectable amounts in a single mature testis from rainbow trout have been purified to homogeneity using acid extraction, gel filtration chromatography on Bio-Gel P-10, ion-exchange chromatography on carboxymethylcellulose and reverse-phase high-pressure liquid chromatography. Each of the six purified protamines was completely sequenced using automated gas-phase Edman degradation. Each protamine is two-thirds arginine and also contains proline, serine, valine and glycine. Three protamines also contain alanine while two contain isoleucine. Four of the protamines have 32 amino acids while the remaining two have 30. The six protamines have been classified into three families on the basis of their amino acid sequences.     

Insulin-stimulating peptide from a tryptic digest of bovine serum albumin: purification and characterization

A procedure was established for isolation of a low molecular weight polypeptide with insulin-stimulating activity in apparent homogeneity from a tryptic digest of bovine serum albumin on a semipreparative scale. Purification of this insulin-stimulating peptide (ISP) was monitored by an adipose-explant assay in which stimulation of fatty acid synthesis from glucose by insulin was measured. The polypeptide was purified by a combination of DEAE-cellulose column chromatography, gel filtration on Bio-Gel P-10, hydrophobic chromatography on a semipreparative C18 reversed-phase HPLC column, and ion exchange chromatography on an SP-5PW HPLC column. The primary structure of ISP was deduced. ISP is a two-chain polypeptide consisting of 71 amino acid residues, and corresponds essentially to residues 115-143 and 144-184 (185) of bovine serum albumin connected to each other by a disulfide bridge. But comparison of the sequence of ISP with that of the relevant regions of bovine serum albumin determined by Brown indicated the presence of one tyrosine insertion between residues 155 and 156 of albumin. Therefore, the molecular weight of ISP was calculated to be 8,496.     

Studies on cytochrome c oxidase, VII. Isolation and chemical characterization of polypeptide VII.

Polypeptide VII of cytochrome c oxidase was isolated and purified by gel filtration on Bio-Gel P-10 in 10% acetic acid. Automatic Edman degradation of this peptide chain was not successful, because it is blocked at the N-terminus. The amino acid analysis shows a relatively high content of hydrophilic residues (54%). On the basis of this analysis and the apparent molecular weight by sodium dodecyl sulfate gel electrophoresis and gel filtration, a chain length of about 80 residues was calculated. Among the tryptic peptides one blocked heptapeptide was found. Cleavage of this peptide with thermolysin gave two peptide fragments, one of which was not retained on a cation exchange resin. Mass spectrometric sequence determination of this peptide revealed the structure Ac-Ala-Glu-Asp for the N-terminus of polypeptide VII. Treatment with carboxypeptidase A at two different pH values showed that the C-terminal amino acid is isoleucine and the penultimate amino acid is lysine.     

Purification and Characterization of the Acidic Lectin from Winged Bean Seeds

Winged bean acidic lectin was purified by DEAE-Sephadex A-50 and affinity chromatography on N-acetylgalactosamine-agarose gel. The purified lectin was a glycoprotein homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and gel filtration. The molecular weight of the lectin was 52,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single component of molecular weight of 27,000. Its isoelectric point was 5.5. The acidic lectin was rich in acidic amino acids, and contained 2mol of methionine but no cystine. It also agglutinated both trypsinized and untreated human erythrocytes (types A, B, AB and O), but not rabbit erythrocytes. The hemagglutination was inhibited by d-galactose and related sugars. Modification of the acidic lectin with N-bromosuccinimide caused a concomitant loss of the hemagglutinating activity with oxidation of tryptophan residue. The acidic lectin was immunologically different from the purified winged bean basic lectin by double immunodiffusion using antiserum raised against the basic lectin.