Studies on Heterogeneous Components of Hog Pancreatic Kallikrein : "Possible Role of the Neuraminic Acid Residues"

抄録

Fucose, mannose, galactose, N-acetylglucosamine and neuraminic acid were detected as structural sugars of the purified hog pancreatic kallikreins A and B. Kallikrein B contained roughly twice as much sugar as kallikrein A (A, 4.56 ; B, 8.40% by weight). Kallikreins A and B were further divided into 3 (A-I, A-II and A-III) and 6 (B-I, B-II, B-III, B-IV, B-V and B-VI) micro-heterogeneous components, respectively. The sugar contents of B-III and B-IV (the main micro-heterogeneous forms of B) were also about twice as much as that of A-II (one of the micro-heterogeneous forms of A). As for the content of neutraminic acid, A (more acidic than B) contained less than B. On the other hand, the contents of neutraminic acid in the micro-heterogeneous components from A and B were different from each other, and the more acidic micro-heterogeneous forms were richer in neuraminic acid. After treatment with neuraminidase, the more acidic microheterogeneous forms shifted to the positions of the less acidic ones. These observations suggested that the neuraminic acid content in the molecule was the main factor permitting the separation of the micro-heterogeneous forms by isoelectric focusing. After the reduction of kallikreins A and B with 2-mercaptoethanol, two protein bands were detected in each case on polyacrylamide gel electrophoresis. However, although staining of the carbohydrate moiety of the reduced B with PAS reagent also showed two bands identical with the protein bands, only one of the protein bands of the reduced A was stained for carbohydrate. Thus, kallikreins A and B both consist of two peptide chains, but one of the peptide chains of A has no carbohydrate moiety, whereas carbohydrate moieties are bound to both peptide chains in the case of kallikrein B. The Km values and optimum pH's of kallikreins A and B, their micro-heterogeneous forms (A-II, A-III, B-IV and B-V) and asialo-kallikreins A and B for Bz-Arg-OEt hydrolysis were closely similar. Asialo kallikreins A and B were less stable than intact sialo-A and -B at 80°during incubation for 1 hr. Thus, neuraminic acid appears not to be related to the enzymatic activity, but dose influence the stability of the enzyme molecules.