Acrosin: immunochemical demonstration of multiple forms generated from bovine and human proacrosin

1983 ◽  
Vol 61 (9) ◽  
pp. 989-995 ◽  
Author(s):  
John S. Elce ◽  
Elise J. McIntyre
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Immunoaffinity Chromatography ◽  
Relative Mass ◽  
Multiple Forms ◽  
Western Blots ◽  
Specific Immunoglobulin ◽  
Single Form ◽  
Bovine Spermatozoa

A rabbit antiserum was prepared against purified bovine spermatozoal acrosin (EC 3.4.21.10), and the specific immunoglobulin G (IgG) was isolated by immunoaffinity chromatography. This IgG was shown to be monospecific for acrosin by rocket immunoelectrophoresis and by Western blotting of sodium dodecyl sulfate – polyacrylamide gels onto nitrocellulose sheets, followed by indirect immunodetection. In extracts of bovine spermatozoa prepared in the presence of 50 mM benzamidine, a single form of acrosin was detected, having a relative mass (Mr) of 48 000, which is presumed to be proacrosin. At least four further intermediate forms of acrosin were detectable in extracts prepared in the absence of benzamidine and in the various column eluates, having Mr values of 47 000, 44 000, 42 000, and 40 000, while the final product of the purification had a Mr of 37 500. The rabbit antibovine acrosin antiserum reacted also with human acrosin on Western blots. In this way, human proacrosin was found to have a Mr of 50 000 and to be convertible into intermediate forms of Mr 48 500, 44 500, 40 500, and 37 500, while the final product had a Mr of 35 500.

Plasma and Platelet Fibrinogen Differ in γ Chain Content

1984 ◽  
Vol 51 (01) ◽  
pp. 084-088 ◽  
Author(s):  
Charles W Francis ◽  
Ralph L Nachman ◽  
Victor J Marder
Keyword(s):  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Immunoaffinity Chromatography ◽  
Plasma Fibrinogen ◽  
Western Blots ◽  
Electrophoretic Mobilities ◽  
Chain Composition ◽  
Normal Human

SummaryThe polypeptide chain composition of fibrinogen and cross- linked fibrin from normal platelets and plasma has been compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fibrinogen was prepared from a lysate of normal human platelets by ethanol precipitation and by antifibrinogen immunoaffinity chromatography. While the Aα chains of platelet fibrinogen appeared to be somewhat degraded, the electrophoretic mobilities of purified platelet and plasma fibrinogen Bβ an γ chains were the same. Crosslinked fibrin was prepared from platelet and plasma fibrinogen and γ chain dimers identified by their electrophoretic mobility, incorporation of the lysine analog dansyl cadaverine during crosslinking, and by reaction with antifibrinogen antibody in Western blots. Platelet crosslinked fibrin had a different polypeptide chain composition than that of plasma crosslinked fibrin with absence of the γ50–γ57.5 dimer in the platelet fibrin. This finding indicates that the γ57.5 chain, which constitutes 5% of total normal plasma fibrinogen γ chains, is either absent or present in markedly reduced amounts in platelet fibrinogen.

Purification Of 1- And 0-Gla Prothrombins And Their Differentiation With Normal And Other Dicoumarol-Induced Prothrombins

1981 ◽  
Author(s):  
O P Malhotra
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Electrophoretic Mobility ◽  
Vitamin K ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
The Other ◽  
Multiple Forms ◽  
Isoelectric Precipitation ◽  
Antigenic Activity

In normal prothrombin, 10-glutamyl residues present in the amino portion of the molecule are carboxylated to form γ-carboxyglutamic acids (gla). Dicoumarol, an antagonist of vitamin K, induces the production of (partially) acarboxylated atypical prothrombins. In addition to our atypical varieties, viz. 7−,5− and 2-gla prothrombins, we have isolated two more atypical molecules, one containing 1.0±0.1 gla (1-gla prothrombin) and the other approximately 0.25 gla (0-gla prothrombin). These two variants adsorbed onto alumina Cγ-gel (Bio-Rad), similar to 2-gla protein, but were derived from 40 to 50% (NH4)2SO4 saturation. The purified materials, obtained after isoelectric precipitation followed by preparatory polyacrylamide-gel electrophoresis and heparin agarose chromatography, showed a single component by analytical disc-gel electrophoresis both in the presence or absence of sodium dodecyl sulfate (SDS) and contained antigenic activity comparable to that of normal prothrombins.The pI’s of the two variants by column electrofocusing were each 4.835±0.015. Similarly, the two proteins did not reveal any difference in electrophoretic mobility; however, their prothrombin fragments 1 (F1, residues 1-156) did—0-gla F1 moved slower than 1-gla F1. Employing anti (normal) prothrombin sera with Ca2+ , the two, 0− and 1-gla, F1’s produced vaguely visible immunoprecipitates which were definitely lighter than all the other F1’s including 2-gla. These results confirm that not only are multiple forms of atypical prothrombin induced by dicoumarol but also that gla does affect the immunochemical properties of the gla-containing fragment.

Schistosoma mansoni: anomalous immunogenic properties of a 27 kDa larval serine protease associated with protective immunity

Parasitology ◽  
1997 ◽  
Vol 115 (3) ◽  
pp. 237-247 ◽  
Author(s):  
H. Y. DARANI ◽  
R. H. C. CURTIS ◽  
C. McNEICE ◽  
H. P. PRICE ◽  
J. R. SAYERS ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Serine Protease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Molecular Size ◽  
Major Constituent ◽  
Partial Purification ◽  
Western Blots ◽  
Sds Page

A cationic Schistosoma mansoni cercarial antigen was shown to be a serine protease as it was capable of hydrolysing N-acetyl-dl-phenylalanine β-naphthyl ester (NAPBNE) after precipitation by immunoelectrophoresis, and this reaction was modulated by the serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP). The antigen in the immunoprecipitin arcs could also be radio-isotope labelled with tritiated DFP. The peptidolytic enzyme identified in immunoelectrophoresis with polyspecific sera and radio-isotope labelled with tritiated DFP had a relative molecular size of approximately 27 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), and evidence obtained after partial purification, SDS–PAGE and immunoblotting supported this size estimate for the enzyme. A rabbit antiserum raised against the peptidolytic antigen reacted against a doublet of antigens at 27/28 kDa in immunoelectrophoresis arcs and against an antigen of 60 kDa in Western immunoblots of crude cercarial homogenate. However, the latter serum precipitated the cationic antigen in immunoelectrophoresed cercarial homogenates only after pre-incubation of the homogenates with PMSF. Fractions containing the partially purified protease also degraded radio-isotope labelled human IgG. The reactivity of a range of polyspecific and monospecific rabbit antisera in Western blots with larval extracts indicated that antibody responses against the 27/28 kDa doublet may be modulated. When immunized with material which contained the 27 kDa enzyme as a major constituent, and which was secreted by S. mansoni cercariae during transformation, only 5 of 16 mice produced antibody to this antigen that was detectable in Western blots. The 5 antibody ‘responder’ mice were significantly (P<0·001) protected against challenge with a percutaneous infection of S. mansoni cercariae compared with a group of mice also immunized with CTF, but which had not produced antibodies against the 27/28 kDa doublet. The results indicate that the 27 kDa serine protease of S. mansoni larvae is a target that is sensitive to immunological attack.

scholarly journals Identity of ligandin in rat testis and liver

1982 ◽  
Vol 203 (1) ◽  
pp. 193-199 ◽  
Author(s):  
K A Eidne ◽  
R E Kirsch
Keyword(s):  
Reduced Glutathione ◽  
Sodium Dodecyl ◽  
Standard Technique ◽  
Electrophoretic Analysis ◽  
Immunoaffinity Chromatography ◽  
Multiple Forms ◽  
Glutathione S Transferase ◽  
Closely Related Species ◽  
Exclusion Chromatography ◽  
Molecular Exclusion Chromatography

1. One of the main problems in the field of multifunctional proteins such as ligandin is the possibility that multiple forms and isoproteins may exist. Because liver ligandin [GSH (reduced glutathione) S-transferase B] consists of equal amounts of Ya (22 000 Da) and Yc (25 000 Da) subunits, and testis ligandin, prepared by the standard technique of anion-exchange and molecular-exclusion chromatography, contains more Yc subunit than Ya, it has been claimed that testis and liver ligandin are different entities. 2. We purified testis ligandin by immunoaffinity chromatography and have obtained a product identical with liver ligandin (Yc = Ya). This suggests that the differences previously described may be due to contamination of testis ligandin by a closely related species. In fact sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of testis GSH S-transferases separated by CM-cellulose chromatography showed that GSH S-transferase AA, present in large amounts, migrated in the same region as Yc subunit. 3. Testis ligandin prepared by the standard technique was similar to that reported [Bhargava, Ohmi, Listowsky & Arias (1980) J. Biol. Chem. 255, 724-727] and contained more Yc subunit than Ya. CM-cellulose chromatography of this ‘pure’ preparation revealed significant amounts of GSH S-transferase AA migrating as Yc subunit, in addition to ligandin consisting of equal amounts of Ya and Yc subunits. 4. Our studies show that testis ligandin is identical with liver ligandin. Previously described differences are due to a contaminant identified as GSH S-transferase AA.

scholarly journals Protein co-isolated with human tissue factor impairs recovery of activity

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 520-523 ◽  
Author(s):  
SD Carson ◽  
SE Ross ◽  
RA Gramzinski
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Tissue Factor ◽  
Human Tissue ◽  
Cyanogen Bromide ◽  
Sodium Dodecyl ◽  
Immunoaffinity Chromatography ◽  
Factor Activity ◽  
Tissue Factor Activity ◽  
Carboxyl Terminal

Abstract Preparations of human tissue factor isolated by immunoaffinity chromatography contain variable amounts of 47,000 mol wt, 55,000 mol wt, and multimeric tissue factor when analyzed without reduction on polyacrylamide gels in sodium dodecyl sulfate (SDS). When analyzed after reduction, the 47,000 mol wt tissue factor apoprotein and a protein of about 12,000 mol wt are observed. Elution of tissue factor from polyacrylamide gel slices, followed by reassociation with lipids, restored proportionately much greater tissue factor activity with the 47,000-mol wt protein than with the 55,000-mol wt form. Cyanogen bromide cleavage at the single tissue factor methionine revealed that the 12,000-mol wt protein is associated with the carboxyl-terminal peptide derived from the 47,000-mol wt protein. These results reveal that association of the 12,000-mol wt protein with the cytoplasmic domain of tissue factor can modulate its activity in vitro.

scholarly journals Extracellular and Cytosolic Iron Superoxide Dismutase from Mycobacterium bovis BCG

1998 ◽  
Vol 5 (6) ◽  
pp. 784-789 ◽  
Author(s):  
Sung-Koo Kang ◽  
Yong-Jae Jung ◽  
Cheorl-Ho Kim ◽  
Chul-Yong Song
Keyword(s):  
Superoxide Dismutase ◽  
Sodium Dodecyl Sulfate ◽  
Mycobacterium Bovis ◽  
Signal Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Western Blots ◽  
Iron Superoxide Dismutase ◽  
Dodecyl Sulfate

ABSTRACT Two forms of iron superoxide dismutase (SOD) were purified from cell extract (CE) and culture filtrate (CF) of Mycobacterium bovis BCG, respectively. The molecular weight of both enzymes was estimated to be approximately 84,000 by gel filtration, whereas that of their subunits was 21,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that each of purified enzymes is composed of four identical subunits. The specific activities of CE SOD and CF SOD were 3,850 and 4,040, respectively. The purified enzymes were not joined by disulfide bonds and were, to some extent, resistant to sodium dodecyl sulfate. Their activities were lost by H2O2, but not by azide and cyanide, indicating iron SODs. Enzyme activities were detectable over a broad range of pHs, from 5.0 to 9.0, and were stable for 6 months at −20°C. Each value of pI was 4.5. In Western blots, both enzymes reacted with sera of tuberculosis patients, but not with normal sera. The N-terminal amino acid sequences of CE SOD and CF SOD were the same, suggesting that there is no N-terminal signal sequence.

Western blots from sodium dodecyl sulfate-polyacrylamide gels stained by metal salts

1989 ◽  
Vol 180 (2) ◽  
pp. 311-313 ◽  
Author(s):  
Ding Wang ◽  
James K. Dzandu ◽  
Mukarram Hussain ◽  
Robert M. Johnson
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Metal Salts ◽  
Polyacrylamide Gels ◽  
Western Blots ◽  
Dodecyl Sulfate
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