Purification and Some Properties of Two Kinds of Keratin-Hydrolyzing Enzymes of Cow Snout Epithelium

Two kinds of keratin-hydrolyzing enzymes (KHEs) from cow snout epithelium were highly purified by affinity chromatography using soybean trypsin inhibitor-bound Sepharose. On gel filtration chromatography, the KHEs were eluted at a volume corresponding to a relative molecular mass (Mr) of 21,000. They were separated from each other by ion exchange chromatography. One of the enzymes had the same characteristics as urea extracted alkaline proteinase, of which optimal pH was at 8.5 to 9.0. Sodium dodecyl Sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single band with Mr of 21,500 in the presence or absence ofa reducing agent. The other enzyme was a neutral proteinase, with an optimal pH of 7.5. Both enzymes were inhibited by phenylmethylsulfonyl fluoride and soybean trypsin inhibitor. Among the fluorogenic peptides that were hydrolyzed most effectively by the alkaline proteinase were peptidyl MCAs (4-methyl-coumaryl-7-amides) with extended sequences, Boc-Leu-Ser-Thr-Arg-MCA, and then Boc-Val-Pro-Arg-MCA. The neutral proteinase hydrolyzed the latter most effectively. They hydrolyzed preferentially high Mr keratins of cow snout and of newborn mouse epidermis, and showed a limited proteolysis toward 68,000 polypeptide, giving rise to distinct products. The high substrate specificity and extended subsites of the KHEs suggest their role on the metabolism of the high Mr keratins

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_ proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer