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91.
An in vitro system for the synthesis of ornithine transcarbamylase (OTCase) was established using iS-30 extract from E. coli MDS6-2(lambda) and DNA of a lambda transducing phage carrying argI and argF genes. This in vitro synthesis was completely dependent on the additon of DNA, and was sensitive to chloramphenicol and rifampicin. Radioisotopic analysis confirmed that the synthesized enzyme catalyzes the carbamylation of ornithine to citrulline. In the in vitro system the repression and derepression of OTCase synthesis could be observed by mixing iS-30 extracts prepared from argR+ and argR- cells. A remarkable maturation effect could be observed for the FFF enzyme, but not for the III enzyme. This system is considered to reflect the in vivo situation, and should therefore be useful for investigations on the regulation of OTCase synthesis in vivo.  相似文献   
92.
δ-Aminolevulinic acid (ALA) synthase was partially purified from liver cytosol fraction of rats treated with allylisopropylacetamide (AIA). The cytosol ALA synthase showed an apparent molecular weight of 320,000. The cytosol ALA synthase of this size dissociates into at least three protein components when subjected to sucrose density gradient centrifugation in the presence of 0.25 m NaCl: one is the catalytically active protein with an s value of about 6.4 or a molecular weight of 110,000, and the other two are catalytically inactive binding proteins showing s values of about 4 and 8, respectively. Recombination of the 6.4 S protein and the 4 S protein yielded a protein complex with an apparent molecular weight of 170,000 and recombination of all three protein components resulted in formation of the original cytosol ALA synthase. The cytosol ALA synthase also loses its binding proteins when treated with various proteases; thus, the enzyme-active protein obtained after papain digestion was very similar, if not identical, to mitochondrial ALA synthase. When treated with trypsin, however, the cytosol ALA synthase was converted to an enzyme showing an apparent molecular weight of 170,000, which probably represents the complex of the mitochondria-type enzyme and the 4 S binding protein. The cytosol ALA synthase tends to aggregate to form a dimer with an apparent molecular weight of 650,000–700,000. The aggregated form of the cytosol ALA synthase was less susceptible to trypsin digestion. Hemin strongly stimulated dimer formation of the cytosol ALA synthase and the aggregate produced by contact with hemin was very tight and did not easily dissociate into its respective protein components by sucrose gradient centrifugation or even after treatment with trypsin. The possible mechanisms of the conversion of cytosol ALA synthase to the mitochondrial enzyme and also of the inhibition by hemin of the intracellular translocation of ALA synthase are discussed.  相似文献   
93.
The levels of free amino acids in the cerebral cortex of acute and chronic uremic rats were examined. Amino acids significantly elevated were aspartate, glutamine, glycine, histidine, ornithine, phenylalanine, phosphoethanolamine and taurine, whereas 1-methyl histidine and 3-methyl histidine were specifically detected in uremic rats. Glutamate, arginine and carnosine disclosed a significant reduction. There was no change in the concentrations of γ-aminobutyrate and alanine. The above findings were essentially identical in both acute and chronic uremia. It was conjectured that these changes of amino acid levels in the brain might participate in the progress of uremic encephalopathy.  相似文献   
94.
A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.  相似文献   
95.
Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.  相似文献   
96.
1H and 15N NMR study of human lysozyme.   总被引:1,自引:0,他引:1  
The 15N signal assignment of human lysozyme was carried out by using 1H-1H and 1H-15N two dimensional experiments. To solve the severe overlap problem of the NH signals, uniform labeling of the protein with 15N was introduced. The uniformly 15N labeled protein was prepared using a high-expression system of Saccharomyces cerevisiae. From the analyses of 1H and 15N NMR spectra, all of the backbone 15N signals of the molecule were assigned to each specific residue in the amino acid sequence. Recently published proton signal assignments [Redfield & Dobson (1990) Biochemistry, 29, 7201-7214] were confirmed by these complementary data. In addition, assignments were extended to side chain 15NH2 groups of asparagine and glutamine. Elements of secondary structure were deduced from the pattern of sequential and medium-range NOE connectivities. Two beta-sheets and four alpha-helices could be identified in the protein, which were in good agreement with those determined by X-ray crystallography. The interaction between human lysozyme and its inhibitor N-acetyl-chitotriose was investigated by 15N-1H HMQC spectra. Most of the 15N-NH cross-peaks in the spectra were separated well enough to be followed during the titration experiment. Residues whose NH proton signals decrease in intensity upon complex formation, are located mainly around subsites B, C, and D. Local conformational changes were observed around the fourth helix adjacent to the cleft of human lysozyme.  相似文献   
97.
The three-dimensional structures of apo- and holomutant human lysozymes (D86/92 lysozyme), in which a calcium binding site was designed and created for enhancing molecular stability by replacing both Gln86 and Ala92 with aspartic acids, were refined at 1.8-A resolution by x-ray crystallography. The overall structures and crystallographic thermal factors of all three proteins, the apo-, holo-D86/92, and the wild-type human lysozymes, were essentially identical; these results showed that the introduction of the calcium binding site did not affect either the overall structure or molecular rigidity of the proteins. However, structure analyses of the apo-D86/92 lysozyme revealed that the mutations affected the side chain conformation of residue 86 and hydrogen networks between the protein and the internal solvent molecules. In the structure of the holo-D86/92 lysozyme, seven oxygen ligands formed a slightly distorted pentagonal bipyramid around the calcium ion, indicating that the coordination around the calcium ion was quite similar to that in baboon alpha-lactalbumin. The pentagonal bipyramid coordination could be one of the most widely found and appropriate calcium binding schemes in proteins.  相似文献   
98.
Signal sequences play a central role in the initial membrane translocation of secretory proteins. Their functions depend on factors such as hydrophobicity and conformation of the signal sequences themselves. However, some characteristics of mature proteins, especially those of the N-terminal region, might also affect the function of the signal sequences. To examine this possibility, several mutants of human lysozyme modified in the N-terminal region of the mature protein were constructed, and their secretion in yeast as well as in vitro translocation into canine pancreatic microsomes were analyzed using an idealized signal sequence L8 (MR(L)8PLAALG). Our results show the following. (1) Change in the charge at the N-terminal residue of the mature protein does not affect secretion drastically. (2) Substitution of a proline residue at the N terminus prevents cleavage of the signal sequence, although translocation itself is not impaired. (3) Excessive positive charges in the N-terminal region delay translocation of the precursor protein across the membrane. (4) Polar and negatively charged residues introduced into the N-terminal region affect the secretion of the mature protein by preventing its correct folding.  相似文献   
99.
A murine mAb, 7D3, was produced by fusion of spleen cells obtained from mice immunized with a rat thymic epithelial cell line, Tu-D3 and NS/1 myeloma cells. 7D3 antibody reacted with approximately 95% thymocytes, 17% spleen cells, less than 9% of mesenteric lymph node cells and 32% of bone marrow cells of rat origin. 7D3 also reacted with two rat thymic epithelial cell lines but not with a rat fibroblastic cell line. Immunochemical analysis demonstrated that 7D3 antibody recognized a single polypeptide with molecular weight of 80,000 in FTE cells and 80,000 to 96,000 in thymocytes. 7D3 antibody strongly inhibited the thymocyte binding to thymic epithelial cells. In addition, 7D3 antibody inhibited TPA-induced thymocyte aggregation. 7D3 negative rat thymic lymphoma cells bound to 7D3 positive thymic epithelial cells and this binding was inhibited by 7D3 antibody, indicating that a part of thymocyte-thymic epithelial cell binding was mediated by the interaction of 7D3 Ag and undefined ligand to 7D3.  相似文献   
100.
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