Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH,-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin 1 was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80°C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine . HCl and 1 YO SDS) at 23 "C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.The enzymes of thermophilic bacteria are generally heatstable [I], and the stability of these proteins is interesting for protein chemistry. Besides their scientific interest, these heatstable enzymes are valuable because of their bioengineering and biotechnological applications. We have studied the enzymes of extremely thermophilic bacteria belonging to the genus Thermus, mainly L-lactate dehydrogenases [2 -51 and proteases [6,7]. Heat-stable proteases are expected to be useful for application [8].At present three Thermus species strains are known which produce heat-stable extracellular proteases. A metal protease (called caldolysin) produced by Thermus T-351 has been purified and characterized [9]. We reported that Thermus caldophilus GK24 produces a neutral serine protease which forms a complex with several kinds of proteins and which appears homogeneous on chromatographic analysis [6]. These two proteases are extremely heat-stable and resistant to denaturing treatments [6, 91. We previously reported the production of two kinds of extracellular proteases, aqualysins I and I1 [7], by Thermus aquaticus YT-1 [lo]. Aqualysin I is an alkaline serine protease, and aqualysin I1 an chelator-sensitive, neutral serine protease, the optimum temperatures for the proteolytic activities of the two enzymes being around 75°C and 95°C respectively [7]. Correspoiidence to H. Matsuzawa, Department of AgriculturalChemistry, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 113Abbreviations. iPr,P-F, diisopropylfluorophosphate; Z-AlaGly-PheCH2C1, benzyloxycarbonyl-Ala-Gly-Phe-chloromethane; CIHgBzOH, p-chloromercuribenzoate; TosPheCH2C1, N-tosylphenylalanylchloromethane; TosLysCH2Cl, Nu-tosyl-L-lysylchloromethane; Suc-Ala-Ala-Ala-NH-Np, succinyl-Ala-Ala-Ala p-nitroanilide; Cya, cysteic acid. Except where otherwise specified, the constituent amino acids were all of the L configuration.Enzymes. Serine proteinase (EC 3.4.21 .-); microbial serine proteinase (EC 3.4.21.14); subtili...
New Zealand Black (NZB) mice spontaneously produce a variety of autoantibodies, including those to nucleic acids, T ceils, and erythrocytes (1, 2), and show a high serum level of IgM that is probably due to the spontaneously occurring polyclonal activation of B cells (3, 4). They also develop immune complex-type glomerulonephritis resembling human lupus nephritis (1). Additional abnormalities found in NZB mice are the productions of a large amount of gp70, a major constituent of C-type retroviral envelope glycoprotein, and the antibodies to gp70, resulting in the formation of gp70 immune complexes (gp70 ICs) (5). These gp70 ICs, as well as DNA-anti-DNA ICs (6), have been implicated in the pathogenesis of renal disease in NZB and their progeny (5, 7-9). All these immunological abnormalities are under the control of multiple genes of NZB mice, and a specific genetic mechanism regulates the expression of each of the various traits (10).As compared with this NZB strain, (NZB × NZW)F1 (B/W F1) hybrids show an earlier onset and a higher incidence of proteinuria associated with increased serum levels ofanti-DNA antibodies, gp70 ICs and IgG (1,9, 11). These findings can be explained by the involvement of a New Zealand White (NZW) gene(s) that acts to intensify the expression of relevant autoimmune NZB gene(s) in B/ W F1 hybrids. Either one or two dominant NZW gene(s) have been implicated in the increased incidence and severity of the renal disease observed in B/W F1 hybrids (9, 12). Maruyama et al. (9) suggested that a single dominant NZW gene acts to intensify the production of anti-gp70 antibodies, which in turn results in the formation of a greater amount of gp70 ICs in B/W F1 hybrids. Our recent studies showed that increments in the serum level of anti-dsDNA antibodies in the F1 hybrids can be attributed to the combined effect of two independently segregating dominant NZW genes. 1 All these genetic studies in B/W F1 × NZB backcrosses revealed that each one of these three traits, the increased severity of renal disease and the enhanced productions of gp70 ICs and anti
The progress of growth of Thermus aquaticus YT-1 and production ofcasein hydrolytic enzyme in the culture supernatant were followed, and it was found that the cells produced at least two kinds of extracellular proteases. The properties of the crude enzymes were examined. One of the
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