SEQUENCE-ANALYSIS AND CHARACTERIZATION OF THE PORPHYROMONAS-GINGIVALIS PRTC GENE, WHICH EXPRESSES A NOVEL COLLAGENASE ACTIVITY
SEQUENCE-ANALYSIS AND CHARACTERIZATION OF THE PORPHYROMONAS-GINGIVALIS PRTC GENE, WHICH EXPRESSES A NOVEL COLLAGENASE ACTIVITY
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DOI：
10.1128/jb.174.12.3889-3895.1992
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发表时间：
1992-06-01
影响因子：
3.2
通讯作者：
KURAMITSU, HK
中科院分区：
文献类型：
--
作者：
KATO, T;TAKAHASHI, N;KURAMITSU, HK
为了研究细菌胶原酶在牙周组织破坏中的潜在作用，我们最近从牙龈卟啉单胞菌ATCC 53977中分离出一个表达胶原酶活性的基因prtC（N.高桥T. Kato和H. K. Kuramitsu，FEMS Microbiol。Lett. 84：135-138，1991）。该基因的核苷酸序列已被确定，推导的氨基酸序列对应于一个37.8 kDa的碱性蛋白。Southern杂交分析表明，prtC基因在牙龈卟啉单胞菌的3种主要血清型中具有保守性。该酶已被纯化到接近同质的大肠杆菌克隆NTS 1后，Mono Q阴离子交换和连续凝胶过滤色谱。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测得纯化酶的分子量约为1.25。35 kDa，凝胶过滤层析后活性酶表现为二聚体。胶原酶降解可溶性和重构的纤维状I型胶原、热变性I型胶原和azocoll，但不降解明胶或合成胶原酶底物4-苯基偶氮苄氧基羰基-Pro-Leu-Gly-Pro-D-Arg。酶活性增强钙离子和抑制EDTA，巯基阻断剂，和唾液肽组胺素。还获得了菌株53977表达的第二种胶原酶存在的初步证据。
In order to examine the potential role of bacterial collagenases in periodontal tissue destruction, we recently isolated a gene, prtC, from Porphyromonas gingivalis ATCC 53977, which expressed collagenase activity (N. Takahashi, T. Kato, and H. K. Kuramitsu, FEMS Microbiol. Lett. 84:135-138, 1991). The nucleotide sequence of the gene has been determined, and the deduced amino acid sequence corresponds to a basic protein of 37.8 kDa. In addition, Southern blot analysis indicated that the prtC gene is conserved among the three major serotypes of P. gingivalis. The enzyme has been purified to near homogeneity from Escherichia coli clone NTS1 following Mono Q anion exchange and sequential gel filtration chromatography. The molecular mass of the purified enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be ca. 35 kDa, and the active enzyme behaved as a dimer following gel filtration chromatography. The collagenase degraded soluble and reconstituted fibrillar type I collagen, heat-denatured type I collagen, and azocoll but not gelatin or the synthetic collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg. Enzyme activity was enhanced by Ca2+ and inhibited by EDTA, sulfhydryl-blocking agents, and the salivary peptide histatin. Preliminary evidence for the existence of a second collagenase expressed by strain 53977 was also obtained.