The subunit and polypeptide structure of hexosaminidases from human placenta

1980 ◽  
Vol 58 (4) ◽  
pp. 287-294 ◽  
Author(s):  
D. Mahuran ◽  
J. A. Lowden
Keyword(s):  
Molecular Weight ◽  
Disulfide Bond ◽  
Hydrophobic Interaction ◽  
Sodium Dodecyl ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Α Subunit ◽  
Chain Dimer ◽  
Α Chain ◽  
Polypeptide Structure

Previous reports have indicated that the hexosaminidases (HEX) are tetrameric enzymes composed of either two dimers of β chains (HEX B) or a β-chain dimer and an α-chain dimer (HEX A). HEX S contains only α chains. The apparent molecular weight of both α and β chains is 25 000. We isolated and purified HEX A and B more than 6000-fold. From HEX A we prepared HEX B and a more anodically migrating HEX S, which contained only α chains. After either extensive reduction and alkylation or performic acid oxidation, HEX B gave only a single protein band in polyacrylamide gel electrophoresis in sodium dodecyl sulfate; HEX A consistently gave two bands indicating it contained chains of two different molecular weights. Other reports note two bands in HEX A but have dismissed the high molecular weight band as a dimer caused by either hydrophobic interaction or an unbroken disulfide bond. To rule out hydrophobic interaction, we determined molecular weight by gel filtration in 6 M guanidine–HC1. To ensure complete disulfide bond breakage we used a harsher reduction and alkylation procedure than previously employed by others. The results were then confirmed by the use of two performic acid oxidation techniques, the stronger method resulting in extensive peptide bond oxidation. Samples of HEX A under reduction and alkylation or the weaker performic acid oxidation procedure, again chromatographed as two approximately equal peaks (50 000 and 25 000). HEX B, and the HEX B prepared from HEX A, resulted in one 25 000-dalton peak. HEX S chromatographed as a single 50 000 peak. We conclude that either the α chain in HEX has a molecular weight of 50 000 with one α chain per α subunit or that two 25 000-dalton chains are united by a nondisulfide crosslink (e.g., an isopeptide bond).

The Half-Molecule of Haptoglobin: Studies on the Product Obtained by the Selective Cleavage of a Haptoglobin Disulfide

1973 ◽  
Vol 51 (3) ◽  
pp. 265-273 ◽  
Author(s):  
B. Malchy ◽  
O. Rorstad ◽  
G. H. Dixon
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Disulfide Bond ◽  
Gel Electrophoresis ◽  
Cyanogen Bromide ◽  
Sodium Sulfite ◽  
Sodium Dodecyl ◽  
Performic Acid ◽  
Cleavage Reaction ◽  
Dodecyl Sulfate

A disulfide of haptoglobin 1-1 has been selectively cleaved using sodium sulfite and p-chloro-mercurisulfonate. The reaction product (half-haptoglobin) could be separated from haptoglobin by gel electrophoresis in urea or sodium dodecyl sulfate, and its molecular weight was one-half that of native haptoglobin 1-1. When the sulfite cleavage reaction was performed on haptoglobin 2-2 the haptoglobin polymers were broken down. Isolation of the S-sulfocysteine-containing peptide revealed that the disulfide bond broken in the formation of half-haptoglobin was the 21α-21α disulfide. This conclusion was confirmed by analysis of the cyanogen bromide fragments from half-haptoglobin and by a performic acid diagonal analysis of peptic peptides obtained from half-haptoglobin.

IMMUNOLOGICAL RELATIONSHIPS AMONG OVINE GLYCOPROTEIN HORMONES

1979 ◽  
Vol 83 (2) ◽  
pp. 149-155 ◽  
Author(s):  
M. R. SAIRAM
Keyword(s):  
Cross Reaction ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Glycoprotein Hormones ◽  
Immunological Reactivity ◽  
Amino Groups ◽  
Α Subunit ◽  
Structural Modifications ◽  
Α Subunits ◽  
Bovine Tsh

An antiserum to partially purified ovine FSH (but essentially free of LH) bound 125I-labelled ovine LH or bovine TSH. The antibody was directed exclusively against determinants in the α subunit. In a radioimmunoassay, only the intact ovine and bovine hormones and their α subunits were reactive; the hormone specific β subunits exhibited no cross-reaction. The antibody directed against the α subunit was highly dependent on conformation. Human LH, FSH, TSH or their α subunits did not cross-react in the radioimmunoassay. Structural modifications such as acylation of the amino groups, reduction and alkylation of the S—S-bridges, or performic acid oxidation of the intact ovine FSH, LH or their α subunits virtually eliminated immunological reactivity. Using the α subunit radioimmunoassay, the presence of a significant quantity of free intact α subunit in standard (NIH) preparations of TSH-B7, FSH-S10 and LH-S19 was demonstrated.

Glyceride-cysteine lipoproteins and secretion by Gram-positive bacteria

1982 ◽  
Vol 152 (1) ◽  
pp. 315-322
Author(s):  
J B Nielsen ◽  
J O Lampen
Keyword(s):  
Outer Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Average Molecular Weight ◽  
Sodium Dodecyl ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Gram Positive ◽  
E Coli ◽  
Periplasmic Proteins ◽  
Functional Equivalent

The membrane penicillinases of Bacillus licheniformis and Bacillus cereus are lipoproteins with N-terminal glyceride thioether modification identical to that of the Escherichia coli outer membrane lipoprotein. They are readily labeled with [3H]palmitate present during exponential growth. At the same time, a few other proteins in each organism become labeled and can be detected by fluorography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total membrane proteins. We distinguish these proteins from the O-acyl proteolipids by demonstrating the formation of glyceryl cysteine sulfone after performic acid oxidation and hydrolysis of the protein. By this criterion, B. licheniformis and B. cereus contain sets of lipoproteins larger in average molecular weight than that of E. coli. Members of the sets probably are under a variety of physiological controls, as indicated by widely differing relative labeling intensity in different media. The set in B. licheniformis shares with membrane penicillinase a sensitivity to release from protoplasts by mild trypsin treatment, which suggests similar orientation on the outside of the membrane. At least one protein is the membrane-bound partner of an extracellular hydrophilic protein, the pair being related as membrane and exopenicillinases are. We propose that the lipoproteins of gram-positive organisms are the functional equivalent of periplasmic proteins in E. coli and other gram-negative bacteria, prevented from release by anchorage to the membrane rather than by a selectively impermeable outer membrane.

MOLECULAR WEIGHT OF BOVINE PLASMA ALBUMIN, VITELLENIN, AND THEIR OXIDATION PRODUCTS IN FORMIC ACID

1961 ◽  
Vol 39 (9) ◽  
pp. 1405-1417 ◽  
Author(s):  
W. G. Martin
Keyword(s):  
Molecular Weight ◽  
Formic Acid ◽  
Transient State ◽  
Oxidation Products ◽  
Performic Acid ◽  
Bond Rupture ◽  
Acid Oxidation ◽  
Plasma Albumin ◽  
Viscosity Measurements ◽  
Bovine Plasma

Sedimentation, diffusion, and Archibald transient state measurements were made on bovine plasma albumin and vitellenin of egg yolk in formic acid (88% w/w) solution. The molecular weight of bovine plasma albumin, averaging 77 × 103and 70 × 103with and without added salt, respectively, indicated that peptide bonds were stable to the acid for at least 1 week (storage at 5 °C and measurement periods at 20 °C). Similar values were obtained from estimates based on viscosity measurements but greater deviations occurred. Vitellenin had a mean molecular weight of 93 × 103from sedimentation and diffusion but polydispersity was revealed by the Archibald measurements (molecular weights from 55 × 103to 10 × 103). Higher values of molecular weight were obtained for vitellenin by varying the dissolution technique and exposure time in formic acid and also when viscosity measurements were used to compute molecular weight. Analyses of N-terminal amino acids showed that peptide bond rupture was not a major factor in the polydispersity of vitellenin. Although aggregates are probably present in formic acid solutions of this protein, it appears to be naturally polydisperse. Both albumin and vitellenin were considerably degraded by performic acid oxidation procedures.

The Activation of Human Factor IX

1975 ◽  
Vol 33 (03) ◽  
pp. 553-563 ◽  
Author(s):  
B Østerud ◽  
K Laake ◽  
H Prydz
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Tissue Thromboplastin ◽  
Native Factor

SummaryThe activation of factor IX purified from human plasma has been studied. Factor XIa and kallikrein separately activated factor IX to factor IXa. In both cases factor IX a had an apparent molecular weight of about 42–45000 in sodium dodecyl sul-phate-polyacrylamide disc gel electrophoresis compared with a molecular weight of about 70000 for the native factor IX. The activation by XIa required Ca2+-ions whereas Ca2+-ions did not influence the activation by kallikrein. A mixture of tissue thromboplastin and factor VII or RusselPs-viper venom alone did not activate factor IX. Trypsin activated and plasmin inactivated factor IX.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

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