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Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75,000, [ring 2-13C]Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CATIII) using a histidine auxotroph of E. coli. Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring. One- and two-dimensional spectra revealed six signals out of a total of seven histidine residues in CATIII. pH titration, chemical modification and ligand binding were used to demonstrate that the signal from H195, the histidine at the active site, is not among those observed. Nevertheless, this work demonstrates that selective isotopic enrichment and multiple quantum coherence techniques can be used to distinguish proton resonances in a protein of high molecular mass. 相似文献
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L Y Lian J P Derrick M J Sutcliffe J C Yang G C Roberts 《Journal of molecular biology》1992,228(4):1219-1234
We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints. A protocol which involved distance geometry, simulated annealing and restrained molecular dynamics was used to determine ensembles of 40 structures consistent with these restraints. The structures are found to consist of an alpha-helix packed against a four-stranded antiparallel-parallel-antiparallel beta-sheet. The structures of the two domains are compared to each other and to the reported structure of a similar domain from a protein G from a different strain of Streptococcus. We conclude that the difference in affinity of domains II and III for IgG is due to local changes in amino acid side-chains, rather than a more extensive change in conformation, suggesting that one or more of the residues which differ between them are directly involved in interaction with IgG. 相似文献
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Localization and synthesis of entactin in seminiferous tubules of mouse testis was studied by immunocytochemistry. Frozen sections from adult mice testes were subjected to anti-entactin and anti-laminin immunofluorescence. Both entactin and laminin were localized within the seminiferous tubule basement membrane and intertubular region of the testis. The addition of excess amount of entactin (but not fibronectin), premixed with anti-entactin antiserum, abolished the immunostain. Western blotting showed that a protein extract from a seminiferous tubule basement membrane preparation was recognized by anti-entactin anti-serum and comigrated with recombinant entactin. Enriched fractions of isolated primary Sertoli cells and peritubular myoid cells cultured for 6 days on a glass coverslip were able to synthesize and secrete entactin as detected by immunofluorescence microscopy. Entactin was also produced by TM3 (Leydig-like) and TM4 (Sertoli-like) cell lines as detected by both immunofluorescence and Western blotting. The distribution of entactin vs. laminin within both the cultured primary cells and the TM3 and TM4 cell lines differed. Entactin appeared mainly localized extracellularly. In contrast, laminin was mainly localized intracellularly. The above findings suggested that 1) entactin existed in the seminiferous tubule basement membrane and intertubular region of adult mice testis, co-localized with laminin; 2) entactin was synthesized by the cultured primary Sertoli cells and peritubular myoid cells and the TM3 and TM4 cell lines; 3) entactin was exocytosed with little intracellular accumulation, in contrast to an intracellular accumulation of laminin. 相似文献
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本文用~(125)Ⅰ标记LC-1进行了一些体内外实验。实验结果表明:LC-1单抗的结合常数为4.8×10~8M~(-1),LC-1针对的SPC-A_1细胞表面抗原的位点数为7.2×10~4/细胞;LC-1与LAC-122两单抗针对的抗原决定簇没有交叉;用蛋白酶和过碘酸钠处理SPC-A_1细胞,前者对LC-1的结合抑制39%,后者抑制66%;LC- 1不但有较强的体外结合靶细胞的能力,从LC-1在荷瘤裸鼠中的组织器官分布来看,LC-1与肿瘤有较高的体内亲和性,并且是特异性的结合。 相似文献
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Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli. 总被引:12,自引:5,他引:7 下载免费PDF全文
The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded. 相似文献
27.
The specialized calcium binding amino acid, γ-carboxyglutamic acid (Gla) is quantitated in developing atherosclerotic plaque relative to progression of the disease, and a Gla-containing protein isolated from calcified atherosclerotic plaque is partially characterized. Low levels of Gla are found in fatty streak and fibrous plaque lesions, and a marked increase in Gla content occurs in calcified plaque. A unique Gla-containing protein is purified from 0.5M EDTA (pH 8.0) extracts of calcified plaque, named atherocalcin. The protein containing 19 Gla residues/1000 amino acids is 80,000 molecular weight, with a pI of 4.16 – 4.3 and is uniquely different from other known Gla-containing proteins. The implications of this work for the further understanding of the pathogenesis and therapy of atherosclerosis are discussed. 相似文献
28.
Robert C. Siegel Jane B. Lian 《Biochemical and biophysical research communications》1975,67(4):1353-1359
To date, collagen appears unique among proteins in that it contains histidine in certain of its cross-links. Synthesis of histidine containing collagen cross-links was studied in vitro with lathyritic L-14C histidine or L-14C lysine labelled bone collagen fibrils and purified lysyl oxidase. Synthesis of the tetrafunctional cross-link, dehydrohistidinohydroxymerodesmosine occurred with lysyl oxidase and was inhibited by β-aminopropionitrile. Synthesis began after a lag period of sixteen hours and then proceeded linearly for four days. These data indicate that enzyme dependent synthesis of dehydrohistidinohydroxymerodesmosine occurs in vitro in a biochemically defined system. Biosynthesis in vivo might occur under similar conditions. 相似文献
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