Isolation and characterization of two thiol proteinase inhibitors of low molecular weight from newborn rat epidermis.
Two low-molecular-weight thiol proteinase inhibitors, TPI(1) and TPI(2), were purified from newborn rat epidermis by homogenization and extraction at 37 degrees C, followed by lyophilization, Sephadex G-75 gel filtration and DEAE-cellulose column chromatography. Both purified inhibitors were shown to be homogeneous by polyacrylamide gel electrophoresis in the presence and absence of SDS. TPI(1) was more anionic than TPI(2), and the molecular weights of TPI(1) and TPI(2) were determined to be ca. 12,000 and 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 X 10(-8) M and 4.57 X 10(-7) M for TPI(1) and TPI(2), respectively, calculated from the BAPA hydrolytic activity of papain. Both inhibitors were quite stable proteins as to heat and extreme pHs. 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 trptophanyl residue. From these results, TPI(1) and TPI(2) seem to be iso-inhibitors. The absorption and CD spectra of TPI(1) and TPI(2) in the near ultraviolet 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 ultraviolet 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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000831:Animals, Newborn; D002942:Circular Dichroism; D005260:Female; D007700:Kinetics; D008970:Molecular Weight; D010206:Papain / Q000037:antagonists & inhibitors; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D012867:Skin / Q000737:chemistry*
D000596:Amino Acids; D011480:Protease Inhibitors; D010206:Papain