Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.     

A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization

In the present studies, we have purified a novel small Mr GTP-binding protein, designated as smg p21, to near homogeneity from bovine brain crude membranes, isolated the complementary DNA (cDNA) of this protein from a bovine brain cDNA library, determined the complete nucleotide and deduced amino acid sequences, and characterized the kinetic properties. The cDNA of smg p21 has an open reading frame encoding a protein of 184 amino acids with a calculated Mr of 20,987. The Mr of purified smg p21 is estimated to be about 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Homology search indicates that smg p21 is a novel protein with the consensus amino acid sequences for GTP/GDP-binding and GTPase domains but shares about 55% amino acid sequence homology with the human c-Ha-ras protein. Moreover, smg p21 has the same putative effector domain as the Ha-, Ki-, and N-ras proteins at the same position and the same consensus C-terminal sequence as in these ras proteins. Consistent with these structural properties, smg p21 binds specifically [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), GTP, and GDP with a Kd value for GTP gamma S of about 40 nM. smg p21 binds about 0.7 mol of GTP gamma S/mol of protein. [35S]GTP gamma S-binding to smg p21 is inhibited by pretreatment with N-ethylmaleimide.smg p21 hydrolyzes GTP to liberate Pi with a turnover number of about 0.007 min-1. These kinetic properties of smg p21 are similar to those of the c-ras proteins. These results suggest that smg p21 is a novel GTP-binding protein exerting action(s) similar or antagonistic to that (those) of the ras proteins.     

EnterohemorrhagicEscherichia coli O157:H7 possesses somatic (O) antigen identical with that ofSalmonella O301

The O antigen of enterohemorrhagicEscherichia coli O157:H7 is identical with that ofSalmonella O30₁ and is also related toSalmonella O30₁30₂ in an a-a, b type of relationship.     

In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit.

Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.     

Unusual solvent effect on protease activity and effective optical resolution of amino acids by hydrolytic reactions in organic solvents

Summary The catalytic activities of -chymotrypsin, subtilisin Carlsberg, and subtilisin BPN' for hydrolysis of amino acid esters in acetonitrile-water were unusually dependent on the solvent composition. The products obtained as precipitates in high concentrations of acetonitrile were L-amino acids of high optical purities, and effective optical resolution of amino acids was achieved.     

Chondrosarcoma of the trachea. A case report and literature review

The cytologic presentation of a case of chondrosarcoma of the trachea in a 72-year-old man is described. A mass detected on routine chest roentgenogram and defined by CT scan was used to make a touch imprint smear during partial tumor resection. The cytologic findings included round or polygonal cells with occasional binucleation, round hyperchromatic nuclei and prominent nucleoli, present in an amorphous pink-violet or light-blue background containing fragments of chondroid tissue. The histopathology was interpreted as a low-grade chondrosarcoma. Cartilaginous tumors of the trachea should be considered in the differential diagnosis of upper airway obstruction.     

Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae

The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined. This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and alpha-chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S-carboxymethylated or S-aminoethylated protein. Digestion with A. lyticus protease I was successfully used to degrade the N-terminal half of the S-aminoethylated protein at cysteine residues. RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29,155. The sugar content is 7.9% (by mass). Three glycosylation sites were determined at Asns 15, 76 and 239. Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1. These similar residues may be important for the catalytic activity of RNase T2.     

Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase cytochrome P-450 from hepatic microsomes

Employing reconstitution assays and measurement of cytochrome P-450 content, lanosterol 14 alpha-demethylase and cholesterol 7 alpha-hydroxylase have been studied in solubilized preparations of rat hepatic microsomes. Both activities have been resolved from other cytochrome P-450 isozymes and each other by chromatography on DEAE-Sephacel and adsorption on hydroxylapatite. The demethylase has been further purified to homogeneity by cation exchange chromatography on Mono-S resin. The purified cytochrome displays a specific content of 15.8 nmol of heme/mg of protein and a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 51,000. A Soret maximum for the reduced/CO binding complex at 448 nm is observed. Reconstitution of the purified cytochrome with NADPH-cytochrome-c reductase, dilaurylphosphatidylcholine, NADPH, and O2 supports the demethylation process which is inhibited by CO. Reconstitution also affords accumulation of oxygenated, metabolic intermediates with single catalytic turnover of the cytochrome, thus supporting the hypothesis that a single isozyme of cytochrome P-450 is responsible for all three oxidations and the lyase activity involved in the lanosterol C-32 demethylation sequence. Low oxidase activity toward several xenobiotic substrates and selectivity toward endogenous sterol substrates is observed for the purified cytochrome. These results indicate a high degree of substrate specificity for the cytochrome, which would be expected for a constitutive P-450 involved in anabolic biochemical processes.     

Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO₃⁻-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg²⁺, a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.