We obtained monoclonal antibodies against protein kinase C subtypes (MC-1a, -2a, and -3a) using protein kinase C as antigen which was purified by original affinity chromatography. These monoclonal antibodies recognized the N-terminal region of protein kinase C. On the other hand, polyclonal antibody was obtained synthetic forty peptides of C-terminal of protein kinase C as antigen.
We developed two sites enzyme immuno-assay using both monoclonal and polyclonal antibodies. This sandwich method was used to determine the content of protein kinase C isozymes in tissues of rabbit.

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Electrophoretic analyses of esterases and superoxide dismutase activities of serum, kidney and liver were carried out in ddY mice and six strains of inbred house mice, BALB/c, C3H/He, DBA/2, B10A, B10, C57 BL/6. Sexual dimorphism of esterase activities was observed in serum and kidney but not in the liver, whereas superoxide dismutase (SOD) activities in serum, kidney and liver were not apparently sex-dependent. Serum esterase isozyme patterns among the six strains of inbred house mice showed four different types of banding, namely, BALB/c and C3H/He; B10A and B10; and DBA/2 and C57 BL/6. The patterns of serum esterase in B10A and B10 mice were converted from male type to female type for 2 weeks and 4 weeks following castration, however the male phenotype was restored by administration of testosterone but not of saline.
The manifestation of trypsin like protease activities were distinct in submaxillary glands of B10A and B10 mice. In B10 male mice, protease activities were continuously reduced during a 4 week-period following castration, and were completely restored by the administration of testosterone for 2 weeks. In B10A male mice, however, the protease activities were reduced by castration within 2 weeks and remained unchanged for another 2 weeks. Moreover protease activities were not significantly increased after the administration of testosterone for 2 weeks. Since B10 and B10A mice are congenic strains, the different responses of these mouse strains suggest that the H-2 locus region on chromosome 17 may be involved in the hormonal regulation of these enzyme activities.

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Mouse submaxillary gland trypsin-like proteases are androgen-dependent enzymes that have been used as marker enzyme for androgen. Mice treated with phenylephrine secreted the trypsin-like proteases into saliva. For collecting saliva, small pieces of cellulose acetate membrane were used as absorbent. Saliva protease activity reflected the gland trypsin-like protease activity. Moreover saliva protease activity showed similar time-course change to the gland protease activity with treatment by castration and by testosterone injection. Use of saliva proteases as marker enzyme for androgen has enabled time-course observations of androgenic effects on individual mice.

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