1. Purification and some properties of two kinds of keratin-hydrolyzing enzymes of cow snout epithelium
F Nakada, S Toku, F Inoue J Invest Dermatol . 1987 Oct;89(4):389-94. doi: 10.1111/1523-1747.ep12471766.
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 of a 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.
2. Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90
I Petropoulos, M Conconi, E Turlin, B Friguet, I Emod, F Biville Biochem J . 1998 Jul 15;333 ( Pt 2)(Pt 2):407-15. doi: 10.1042/bj3330407.
Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.
3. A new fluorogenic peptide substrate for vitamin K-dependent blood coagulation factor, bovine protein C
T Morita, S Sakakibara, H Kato, K Takada, J Stenflo, S Iwanaga, Y Ohno J Biochem . 1981 Nov;90(5):1387-95. doi: 10.1093/oxfordjournals.jbchem.a133604.
Protein C is a precursor of plasma serine proteinases, and its active form inactivates specifically blood coagulation Factor V and Factor VIII. Since a specific and sensitive synthetic substrate for the activated protein C was not known, we studied its amidolytic activity toward 25 fluorogenic peptides of the type peptidyl-4-methylcoumaryl-7-amide (peptidyl MCA). The activated protein C, namely, bovine protein C activated by bovine alpha-thrombin, showed the highest activity toward Boc-Leu-Ser-Thr-Arg-MCA. The enzyme's Km and Kcat values for this substrate were calculated to be 3.3 x 10(-4) M and 8.4 s-1, respectively. Optimum conditions for measurement of activated protein C activity were studied with this substrate. Optimum pH was 8.5. For the maximum activity at pH 8.5, concentrations of 0.1 M NaCl and 1 mM CaCl2 had to be maintained in the reaction mixture. The fluorogenic peptide Boc-Leu-Ser-Thr-Arg-MCA was successfully applied to a simple and accurate assay of protein C during its purification.
