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Isolation and characterization of 2 thiol proteinase inhibitors of low molecular weight from new born rat epidermis

, : Isolation and characterization of 2 thiol proteinase inhibitors of low molecular weight from new born rat epidermis. Journal of Biochemistry (Tokyo) 94(3): 811-820

Two low-MW thiol proteinase inhibitors, TPI(1) and TPI(2), were purified from newborn rat epidermis by homogenization and extraction at C, followed by lyophilization, Sephadex G-75 gel filtration and DEAE-cellulose column chromatography. Both purified inhibitors were homogeneous on polyacrylamide gel electrophoresis in the presence and absence of SDS [sodium dodecyl sulfate]. TPI(1) was more anionic than TPI(2), and the MW of TPI(1) and TPI(2) was .apprx. 12,000 and .apprx. 13,000, respectively. They were highly specific for thiol proteinase; TPI(1) was especially more effective than TPI(2) on the proteolytic activity of casein and hydrolytic activity of papain on authentic substrates, and a similar but lesser effect was detected for ficin. They inhibited the enzyme in a noncompetitive manner with inhibition constants of 6.01 .times. 10-8 M and 4.57 .times. 10-7 M for TPI(1) and TPI(2), respectively, calculated from the BAPA [.alpha.-N-benzoyl-DL-arginine-4-nitroanilide] hydrolytic activity of papain. Both inhibitors were quite stable proteins as to heat and extreme pH. The amino acid composition of TPI(1) and TPI(2) were slightly different from each other, and both inhibitors contained no cysteinyl or cytinyl residue and tryptophanyl residue. TPI(1) and TPI(2) seem to be isoinhibitors. The absorption and CD [circular dichroism] spectra of TPI(1) and TPI(2) in the near UV region indicated that the conformation in the vicinity of aromatic amino acid residues in TPI(1) was slightly different from that of TPI(2). The CD spectra of both inhibitors in the far UV region showed the presence of the .beta. turn structure in the protein moiety, suggesting some important roles of the .beta. turn of both inhibitors in the interaction with the thiol enzymes.


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