Kazuko Akiyama

The chymotrypsin-like activity of human prostate-specific antigen, γ-seminoprotein

γ‐Seminoprotein (γ‐Sm) is a human prostate‐specific antigen and a serine protease judging from the complete amino acid sequence which shows extensive homology with the kallikrein family. The enzymatic activity of γ‐Sm was defined as a chymotypsin‐like activity using reduced and S‐3‐(trimethylated amino)propylated lysozyme and insulin‐oxidized A and B chains as substrates. The ‐Leu Ser‐ peptide bond of lysozyme was rapidly hydrolyzed by γ‐Sm. γ‐Sm also hydrolyzed the ‐Phe Glu‐ of lysozyme and the ‐Leu Cys(SO3H) ‐of insulin B chain. Insulin A chain and arginyl‐ or lysyl‐linkage of these proteins were not hydrolyzed by γ‐Sm at all.

Isolation of a new actin-binding protein from human seminal plasma.

Interaction of a protein of human seminal plasma with actin was detected by agar gel immunoelectrophoresis. A major actin-binding protein was isolated from human seminal plasma using an actin-Sepharose 4B column followed by fast-performance liquid chromatography with an anion-exchange Mono-Q column. The protein showed a single band under reduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a position corresponding to a molecular mass of 20 kDa. This 20 kDa polypeptide was detected in saliva and extracts of the submandibular gland and seminal vesicles as well as seminal plasma by the method of immunoblotting using monospecific antibody against the 20 kDa antigenic component of human seminal plasma. The protein might be called secretory actin-binding protein (SABP).

Quantitative analysis of Lea and Leb antigens in human saliva

We have measured the H type 1, Lea and Leb antigens in the saliva from 129 Japanese individuals by a time‐resolved europium ion fluorometric immunoassay using artificial antigen‐albumin complexes as the reference substances. We confirmed that the amount of Leb was larger than that of Lea in the saliva from secretors (Lea–b+) and vice versa in the saliva from nonsecretors (Lea+b–). Unexpectedly, we discovered appreciable amounts of Leb with small amounts of H type 1 in the saliva from the nonsecretors. The concentration of Leb was about 10, 6 and 35% of the concentration of the Lea in the saliva from the nonsecretors of the A, B and O groups, respectively. The possible formation of Leb from Lea, in addition to the formation of Leb from H type 1, in the salivary glands is discussed.

Measuring H type 1 and H type 2 antigens in human saliva by immunoassay using artificial antigens as standard substances

A time-resolved fluorometric immunoassay for water-soluble H antigens of ABH blood group substances has been developed. Introducing artificial antigens (trisaccharide-albumin complexes for H type 1 and H type 2) as reference substances, H blood group substances in human saliva were measured. The monoclonal antibody, anti-H 1E3, reacted with both H type 1 and type 2 chains, and the commercially available anti-H reacted with H type 2 chain. Using these two antibodies we found 10-20-fold higher concentration of H type 1 compared with that of H type 2 in human saliva. The H type 2 was not found in the saliva samples from nonsecretors, irrespective of ABO phenotypes. The results suggest that approximately 90% of A and B blood group substances in human saliva are built on type 1 chains.

Characterization of a monoclonal antibody specific for H type 2 structure of ABO blood group, and its use for measuring H type 2 on human red blood cells

A monoclonal antibody 3A5 specific for the human red blood cells was produced by immunizing BALB/c mouse with human erythrocyte membranes of group O following the immunization protocol of selectively killing the antigen‐stimulated lymphocytes. The monoclonal antibody 3A5 we obtained agglutinated red blood cells regardless of individuals of blood group A, B, or O types, but not those from a person with a rare para‐Bombay type which is the H‐deficient phenotype. The hemagglutination reaction of 3A5 was not inhibited by saliva from either secretor or nonsecretor individuals. The specificity of 3A5 was studied by adsorption with and elution from synthetic oligosaccharide immunoadsorbents including H disaccharide, and H type 1, 2, 3, and 4 structures. Also, the reactivity of 3A5 with synthetic oligosaccharide‐BSA complexes for H type 1, H type 2, Lea, Leb, A, and B was determined by an immunoassay. We found that 3A5 did not react with any of these synthetic oligosaccharides except H type 2. From these results, the antigenic epitope recognized by 3A5 was demonstrated to be the H type 2 structure. Additionally, the H type 2 substance on erythrocytes was quantitatively analyzed using 3A5.