Purification and partial characterization of thiol protease inhibitors from embryos of the brine shrimp Artemia

• Published in: Biochemistry and cell biology Vol. 70; no. 10-11; p. 1020
• Main Authors: Warner, A H, Sonnenfeld-Karcz, M J
• Format: Journal Article
• Language: English
• Published: Canada 01.10.1992
• Subjects: Amino Acid Sequence; Animals; Artemia - chemistry; Artemia - embryology; Cathepsin B - antagonists & inhibitors; Chromatography, Liquid; Electrophoresis; Humans; Molecular Sequence Data; Molecular Weight; Protease Inhibitors - isolation & purification; Sequence Homology, Amino Acid
• ISSN: 0829-8211
• DOI: 10.1139/o92-146

Thiol protease inhibitor (TPI) proteins in embryos of the brine shrimp Artemia were purified to apparent homogeneity and several of their properties were studied. Four protein fractions containing thiol protease inhibitor activity were obtained by high performance liquid chromatography of Artemia embryo proteins on a C-18 reverse-phase column and these were designated as TPI-1a, -1b, -2, and -3. Acrylamide gel electrophoresis showed that TPI-1a and TPI-1b each consisted of two bands of 11.8 and 13.6 kilodaltons (kDa), while TPI-2 and TPI-3 consisted of only one band of 12.5 kDa. Isoelectric focusing experiments demonstrated that TPI-3 contained one band at pH 5.3, while both TPI-1b and TPI-2 yielded bands at pH 5.2 and 5.3. TPI-1a did not yield any major bands. Amino acid composition analyses of the Artemia TPI proteins showed them to be remarkably similar to one another. All were rich in valine and aspartic and glutamic acids, and devoid of cysteine. Partial trypsin digestion of TPI-1b, TPI-2, and TPI-3 yielded several peptides with identical mobilities on a reverse-phase column and several other peptides with different mobilities, suggesting that the multiple forms of Artemia TPIs may have originated from the same parental protein. N-terminal amino acid sequence analyses of TPI-3 suggest that Artemia TPI proteins are members of the type I cystatin family of protease inhibitors.