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Biomedical subjects

M S Blake

Publications and source records attributed to M S Blake.

61 records · Page 4Linked to original sources
PubMed1984

A surface receptor specific for human IgA on group B streptococci possessing the Ibc protein antigen.

A number of group B streptococcal strains of various serotypes, Ia, Ib, Ic, II, and III were examined for their ability to bind human IgG and IgA. No strains of group B streptococci were found to bind IgG, but many strains possessing the Ibc protein antigen(s) were found to bind a significant amount of IgA. The extent of IgA binding correlated with the amount of a 130,000 mol wt, detergent-extractable protein, and reactivity with the Ic typing sera. Using nitrocellulose blots, it was found that the 130,000 mol wt protein bound human IgA. A method was developed to purify the protein while retaining its ability to bind human IgA. Using solid phase radioimmunoassays, it was determined that the protein bound to the Fc region of monomeric or polymeric IgA and that it failed to bind IgM or any IgG isotype.

Antibody Specificity↗
PubMed1984

Purification and partial characterization of the opacity-associated proteins of Neisseria gonorrhoeae.

Gonococci, grown on agar, frequently give rise to opaque colonies. This opacity phenotype is associated with the presence of one or more outer membrane proteins of approximately 28,000 mol weight. These proteins are included within a class of proteins named proteins II. A method is described to isolate and purify the opacity-associated proteins from Neisseria gonorrhoeae. This method uses high concentrations of calcium and a zwitterionic detergent at pH 4.0. Under these conditions proteins II are readily solubilized from the outer membrane. Further purification is achieved by ion exchange and molecular sieve chromatography in the presence of the zwitterionic detergent. The opacity-associated proteins are very basic with isoelectric points varying between 9.0 to 10.0. Further evidence for their basic nature is their behavior on ion exchange chromatography and their amino acid composition.

Amino Acid Sequence↗
PubMed1984

A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.

A rapid, sensitive method has been developed to detect antibody-antigen complexes on "Western blots." The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which had been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.

Alkaline Phosphatase↗
PubMed1982

Purification and partial characterization of the major outer membrane protein of Neisseria gonorrhoeae.

A procedure is described to isolate the major outer membrane protein (protein I) from Neisseria gonorrhoeae in large quantities. The method involves precipitation of protein I by hexadecyltrimethylammonium bromide (CTB) at low ionic strength. CTB is lethal for the gonococci and solubilizes most other proteins. Protein I is brought into solution by raising the ionic strength, and the nucleic acids are subsequently removed by 20% ethanol precipitation. The CTB is removed by precipitating protein I with ethanol and replaced by N-tetradecyl-N,N-dimethyl-3-ammonia-1-propanesulfonate, a dipolar ionic detergent. Further purification is accomplished by ion-exchange and molecular sieve chromatography. Two species of protein I (34,000 daltons [34K] and 32K) were purified by these methods. The purified proteins reacted with antisera prepared against the homologous organisms. The 34K proteins I generated proteolytic fragments upon treatment with trypsin and chymotrypsin similar to those generated by 34K protein in intact gonococci. The amino acid compositions of the three proteins were much like those of other major proteins of gram-negative organisms.

Amino Acid Sequence↗
PubMed1981

Effects of proteolytic enzymes on the outer membrane proteins of Neisseria gonorrhoeae.

Proteolytic enzymes inhibit the growth of some strains and opacity variants of Neisseria gonorrhoeae. To understand the inhibitory effects of these enzymes, we examined several strains to determine the actions of proteases on the three predominant proteins in gonococcal outer membranes. namely, the major outer membrane protein (protein I), the sometimes-expressed opaque protein (protein II), and protein III. In a comparison of the protein I species expressed by different strains, we observed a pattern based on subunit molecular weight and susceptibility to enzymatic degradation. Protein I species having molecular weights of 34,000 were more susceptible to proteolysis, whereas protein I species having molecular weights of 33,000 were less susceptible, and protein I species having molecular weights of 32,000 were resistant. This pattern was observed both in intact cells and in purified outer membranes. All of the enzymes degraded protein II, but this susceptibility appeared to be influenced in part by the species of protein I present. Protein III was resistant to all of the proteolytic enzymes tested. Based on the resulting fragments from each proteolytic cleavage of proteins I and II and their membrane associations, we suggest how these proteins may be arranged in intact membranes. Our data suggested the presence of an endogenous gonococcal enzyme. This enzyme appeared to degrade proteins I and II into fragments resembling the fragments resulting from the action of chymotrypsin.

Bacterial Proteins↗
PubMed1978

Studies on gonococcus infection. XVI. Purification of Neisseria gonorrhoeae immunoglobulin A1 protease.

A protease which cleaves human immunoglobulin A1 (IgA1) has been purified from broth cultures of Neisseria gonorrhoeae. This IgA1 protease is produced by pilated and nonpilated gonococci throughout their growth cycles. A combination of ammonium sulfate precipitation, column chromatography, and either isoelectric focusing or affinity chromatography was utilized to obtain an enzyme preparation that showed approximately 3,800-fold purification and exhibited two bands (65,000 and 70,000 daltons) by analytical polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate and reducing conditions. IgA1 protease activity is dependent on divalent cations and is heat labile. Detection and quantitation of IgA protease activity utilized an assay in which [125I]IgA1 is incubated with protease preparations and the cleavage products are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Female↗