Yasushi Kasahara

Human serum dipeptidyl peptidase IV (DPPIV) and its unique properties.

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) has been purified 18,000‐fold in a yield of 2.2% from human serum. Serum DPPIV, a serine enzyme with an apparent mass of 250 kDa, consists of two identical subunits with an apparent mass of 100 kDa and is inhibited by DPPIV‐specific inhibitor Diprotin A and also by p‐chloromercuribenzoate (p‐CMB), 2‐mercaptoethanol, HgCl2, CdCl2, SrCl2, and ZnCl2. One of the remarkable properties of DPPIV is that its activity is greatly enhanced by Gly‐X (X: especially, Gly, Gln, Glu and Ser) dipeptides. Gly‐X dipeptides increase not only an apparent Km of serum DPPIV for glycyl‐L‐proline 3,5‐dibromo‐4‐hydroxyanilide nearly 10‐fold, but also an apparent kcat nearly 4‐fold. This mechanism is unclear, but one possibility is that Gly‐Pro from substrate might bind amino acids or dipeptides instead of water molecules as DPPIV transpeptidyl activity reported previously. Another remarkable property of DPPIV is the ability to bind adenosine deaminase‐I and ‐II, as is the case with recombinant soluble CD26 (rsCD26). This probably indicates that DPPIV purified from human serum by our method originates from T‐lymphocytes.

Simple devices and their possible application in clinical laboratory downsizing.

The pressure to contain medical costs prompted the development of sophisticated downsizing options for the clinical laboratory, such as automation, information systems, or miniaturization of analytical methods. Faced with an array of approaches to choose from, medical institutions have to select the method that can best serve their particular purposes and circumstances. However, the concept of downsizing needs to be revised and applied to the entire medical system. One way to reduce total medical expenditure would be to shift to near-patient or POC testing, through the adoption of simple devices for immunoassay based on immunochromatography. Recent advancements in the area of simple devices have suggested their possible application for emergency testing sites. This paper reviews the specific situation of Japan with respect to laboratory downsizing and market environment, and discusses the possible future applications of simple devices to near-patient or POC testing.

Homogeneous enzyme immunoassay for macromolecular antigens using avidin biotin enzyme

A unique homogeneous enzyme immunoassay used for the determination of haptens and macromolecular antigens has been developed. It consists of avidin–antigen conjugate, anti‐ligand antibody, and biotin enzyme (propionyl‐CoA carboxylase). The immunological reaction of the conjugate with antibodies specific to ligand sterically prevents enzyme inactivation by avidin. Goat IgG and theophylline were used as model antigens. Avidin–theophylline conjugate inhibited 100% of enzyme activity. Avidin goat IgG conjugate inhibited 90% of enzyme activity.