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11.
Rubrophilin, a unique brain specific polypeptide, was purified to apparent homogeneity from microsomal fractions of bovine brains. The peptide stains pink with Coomassie Brilliant Blue R-250 (C.I. No. 42660) under specific conditions, has an apparent Mr of 53,000, and is acidic with an apparent pI of 4.9. The purification involves initial solubilization of delipidated microsomes in sodium dodecyl sulfate, followed by ammonium sulfate fractionation, reversed ammonium sulfate gradient elution from diatomaceous earth, gel filtration on polyacrylamide (Biogel P-200), gradient elution chromatography from hydroxylapatite, and reverse-phase chromatography from phenyl-Sepharose. A yield of about 5 mg of rubrophilin was obtained from 9 g of microsomal proteins. Amino acid analysis shows that rubrophilin contains only nine amino acids with residues/mol as follows: alanine (102), glutamic acid (97), lysine (65), proline (55), aspartic acid (48), glycine (44), serine (37), threonine (35), and valine (10). Cysteine, methionine, tryptophan, tyrosine, isoleucine, phenylalanine, histidine, and arginine could not be detected. Relative rubrophilin content of vertebrate brains was as follows: mammals greater than birds greater than reptiles greater than fishes. It is present in mouse retina and human neuroblastoma cell cultures but could not be detected in octopus optic lobe or in cultured C-6 rat glioma cells. 相似文献
12.
A D Blake R A Mumford H V Strout E E Slater C D Strader 《Biochemical and biophysical research communications》1987,147(1):168-173
Desensitization of the beta-adrenergic receptor has been correlated in some cell systems with receptor phosphorylation. Various kinases have been implicated in these phosphorylation processes, including both cAMP-dependent protein kinase and protein kinase C. In the present study, we have utilized the protein sequence information obtained from the cloning of the mammalian beta-adrenergic receptor to prepare synthetic peptides corresponding to regions of the receptor which would be predicted to act as possible substrates for these kinases in vivo. Two of these receptor-derived peptides were found to serve as substrates for these protein kinases. A peptide corresponding to amino acids 257-264 of the beta-receptor is the preferred substrate for the cAMP-dependent protein kinase, while protein kinase C showed a marked preference for phosphorylation of a peptide corresponding to residues 341-351 of the beta-adrenergic receptor. 相似文献
13.
14.
Cloning of the 3'-phosphoadenylyl sulfate reductase and sulfite reductase genes from Escherichia coli K-12 总被引:1,自引:0,他引:1
The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation. pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants. Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5). The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter. The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene. Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant. The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E. coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase. Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively. 相似文献
15.
T T Herskovits P A Blake J A Gonzalez M G Hamilton J S Wall 《Comparative biochemistry and physiology. B, Comparative biochemistry》1989,94(2):415-421
1. The hemocyanins of the Melongenidae family of marine gastropods: Melongena corona, Busycon canaliculatum, B. carica, B. contrarium, and B. spiratum exist in solution as multi-decameric aggregates characterized by sedimentation coefficients of approximately 105 S, 130 S, 150 S, 170 S, and higher values, corresponding to di-, tri-, tetra-, penta-, and larger multi-decameric particles. 2. The hemocyanins of B. contrarium and B. carica seem to form the largest decameric aggregates with the tri- to penta-decamers respresenting the major constitutents. Scanning transmission electron microscopy (STEM), both of unstained, freeze-dried and negatively-stained specimens, shows the presence of discrete aggregates consisting of up to ten decameric units. 3. The particle masses as determined by STEM mass measurements for individual molecules gave integral multiples of from 4.2 x 10(6) to 4.4 x 10(6) daltons ranging from about 8.2 x 10(6) daltons for the typical di-decamer of B. canaliculatum hemocyanin to as high as about 39 x 10(6) and 43 x 10(6) for the nano-and deca-decamers of B. contrarium hemocyanin. 4. The appearance of the higher multi-decamers in both negatively-stained and freeze-dried specimens suggest that they are formed by the addition of decameric units to a single di-decameric unit "tail-wise" in both directions. The higher aggregates formed seem to terminate with a closed head or collar at both ends of the assembly. 相似文献
16.
Monohydroxylation of phenol and 2,5-dichlorophenol by toluene dioxygenase in Pseudomonas putida F1. 总被引:7,自引:6,他引:1
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Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water. 相似文献
17.
A general method is presented for the determination of the KM and Vmax for a nonchromogenic substrate in an experimental system where a chromogenic and a nonchromogenic substrate compete for the active site of a single enzyme. Entire progress curves of absorbance versus time for the transformation of the chromogenic substrate must be obtained in the absence and presence of the nonchromgenic substrate. Two quantities may then be extracted from the entire kinetic curves: the value of a delta Area, the area bounded by the kinetic traces of absorbance versus time in the presence and absence of the nonchromogenic substrate; and the value of delta t(5%), the time required to transform 5% of the chromogenic substrate in the presence of the nonchromogenic substrate minus the corresponding time required in its absence. The values of KM and Vmax for the nonchromogenic substrate may be obtained from the dependencies of delta Area and delta t(5%) upon the concentration of the nonchromogenic substrate. The ability of this procedure to yield the correct values of KM and Vmax was demonstrated using beta-lactamase, beta-galactosidase, alkaline phosphatase, and 19 chromogenic/nonchromogenic substrate pairs. This method is equally valid in the absence or presence of competitive product inhibition and should be applicable to any enzyme-catalyzed, strongly exergonic reaction. 相似文献
18.
Effects of acetylene on hydrogenases from the sulfate reducing and methanogenic bacteria 总被引:1,自引:0,他引:1
S H He S B Woo D V DerVartanian J Le Gall H D Peck 《Biochemical and biophysical research communications》1989,161(1):127-133
The effect of acetylene on the activity of the three types of hydrogenase from the anaerobic sulfate reducing bacteria has been investigated. The (Fe) hydrogenase is resistant to inhibition by acetylene while the nickel-containing hydrogenases are inhibited by acetylene with the (NiFe) hydrogenase being 10-50 fold more sensitive than the (NiFeSe) hydrogenase. In addition the Ni(III) EPR signal (g approximately 2.3) of the "as isolated" (NiFe) hydrogenase was significantly decreased in intensity upon exposure to acetylene. 相似文献
19.
Nine low copy number genomic DNA clones, a ribosomal sequence, and seven cDNA clones were found to identify polymorphisms in cultivated barley (Hordeum vulgare L.). An F2 population consisting of 100 plants was produced from a cross between a high-yielding two-rowed feed barley cultivar and a genetic marker stock homozygous for nine recessive and one dominant morphological marker genes. Through the use of these 10 well-distributed marker genes, five previously mapped isozyme loci, and two storage-protein loci, the approximate recombinational location for each of 17 restriction fragment length polymorphism loci was estimated. One clone, pMSU21, identified variation that appeared to be the result of a small insertion-deletion event that differentiated two-rowed and six-rowed genotypes. This difference was characterized, and one allele was sequenced. Oligonucleotide primers that flanked the insertion-deletion event were synthesized, and DNA samples from the F2 population were subjected to polymerase chain reaction sequence amplification. The variation identified by this technique was determined to be allelic to the variation identified using pMSU21 in Southern blot analysis. Maps of previously undescribed informative clones are included. 相似文献
20.
J C Blake D Sprengers P Grech P A McCormick N McIntyre A K Burroughs 《BMJ (Clinical research ed.)》1990,301(6742):12-15
OBJECTIVE--To determine the frequency of an abnormal bleeding time in patients with cirrhosis and to relate this to known factors that affect primary haemostasis and to the severity of liver disease. DESIGN--Prospective clinical and laboratory study in patients admitted for complications or investigations of liver disease. SETTING--Royal Free Hospital hepatobiliary and liver transplantation unit. SUBJECTS--100 Consecutive inpatients aged 17-74 with various forms of cirrhosis, including alcoholic, biliary, autoimmune, viral, and cryptogenic. At least 10 days had elapsed since any episodes of bleeding, resolution of sepsis, or alcohol intake. No patient was taking any drug known to affect primary haemostasis. MAIN OUTCOME MEASURES--Bleeding time as measured with the Simplate double blade template device. A bleeding time longer than 10 minutes was considered abnormal. Other measures were platelet count, prothrombin time, partial thromboplastin time, packed cell volume, and blood urea, serum bilirubin, and serum albumin concentrations, all measured on each subject at the same time by standard laboratory methods. RESULTS--A weak but significant correlation existed between the bleeding time and the platelet count (rs = 0.483; p less than 0.001). There were significantly lower platelet counts, longer prothrombin times, and higher blood urea and serum bilirubin concentrations in the 42 patients with bleeding times of 10 minutes or more compared with the 58 patients with bleeding times less than 10 minutes. Multiple linear regression analysis showed that the bilirubin concentration as well as the platelet count was independently correlated with the bleeding time. The combination of a platelet count greater than 80 x 10(9)/l and a prothrombin time less than 17 seconds (usually taken as safe limits for performing routine liver biopsy) did not predict a normal bleeding time. Ten of 39 patients fulfilling these criteria had a prolonged bleeding time. CONCLUSIONS--Prolonged bleeding time is common in patients with cirrhosis, even in those with prothrombin times and platelet counts within "safe limits" for invasive procedures. The severity of liver disease as assessed by the bilirubin concentration plays an important part in determining the bleeding time in cirrhosis. The bleeding time should be measured when assessing patients for invasive procedures who have a raised bilirubin concentration or poor hepatic function, even if the platelet count and prothrombin time are considered adequate. 相似文献