Abstract
A thermostable, acid proteolytic activity has been found to be associated with the cells and in the culture medium of Sulfolobus acidocaldarius, an archaebacterium. This acid protease, which has been named thermopsin, was purified to homogeneity from the culture medium by a five-step procedure including column chromatographies on DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Sephadex G-100, monoQ (fast protein liquid chromatography), and gel filtration (high pressure liquid chromatography). The purified thermopsin produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the proteolytic activity was associated with the band. Thermopsin is a single-chain protein as indicated by gel electrophoresis and by a single NH2-terminal sequence. It has maximal proteolytic activity at pH 2 and 90 degrees C. A genomic library of S. acidocaldarius was prepared and screened by an oligonucleotide probe designed from the NH2-terminal sequence of thermopsin. Five positive clones were isolated. From these clones the thermopsin gene was mapped and sequenced. The nucleotide sequence showed that the thermopsin structure is encoded in 1020 bases. In the deduced protein sequence, there are 41 amino acid residues (including the initiation Met) preceding the NH2-terminal position of thermopsin. Most of these residues appear to be characteristic of a leader sequence. However, the presence in this region of a short pro sequence cannot be ruled out. Thermopsin contains a single cysteine at residue 237 that is not essential for activity (Fusek, M., Lin, X.-L., Tang, J. (1990) J. Biol. Chem. 265, 1496-1501. Thermopsin has no apparent sequence similarity to aspartic proteases of the pepsin family nor to pepstatin-insensitive acid protease (Maita, T., Nagata, S., Matsuda, G., Murata, S., Oda, K., Murao, S., and Tsura, D. (1984) J. Biochem. 95, 465-475) and thus may represent a new class of acid proteases. Also absent is the characteristic active site aspartyl sequence of aspartic proteases. There are 11 potential N-glycosylation sites on each thermopsin molecule. The molecular weight estimated from gel filtration (45,000) is larger than that calculated from the sequence (32,651), suggesting that thermopsin is the sequence (32,651), suggesting that thermopsin is glycosylated at at least some of these 11 sites.
Highlights
From the *Laboratory of Protein Studies, Oklahoma Medical Research Foundation and the $§Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
The purified thermopsin produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the proteolytic activity was associated with the band
Because the structure-function relationships of acid proteases was of interest to us, we searched for a thermostable acid protease in an archaebacterium, Sulfolobus acidocaldarius, since this bacterium optimally grows in pH 2 at about 70 “C. In this paper, we report the presence of a thermostable acid protease in S
Summary
I contains data on the proteolytic activity of the culture medium and recovered cells of S. acidocaldarius, measured at 80 “C and pH 3.2. Thermostable proteolytic activity of thermopsin was clearly present in both fractions. The activity in the cell fraction, appeared to be tightly associated with cellular structure (data not shown) and was more difficult to purify. Procedures,” part of “Results” Tables I and II, and Figs. The unmarked arrows represent the sequencing from exo- and mung bean nuclease deletion clones
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