Tissue-specific expression of a novel GTP-binding protein (smg p25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC-12 cells

We have purified a novel GTP-binding protein, designated as the smg-25A protein (smg p25A), from bovine brain membranes and determined its primary structure. In the present studies, the smg-25A mRNA levels in various tissues have been studied. The 1.6-kilobase smg-25A mRNA is detected in rat brain by Northern blot analysis. This mRNA is not detected in other rat tissues including thymus, lung, heart, liver, small intestine, kidney, and skeletal muscle. The 1.6-kilobase smg-25A mRNA is also detected in bovine adrenal medulla but not in the cortex. Moreover, this mRNA is detected in rat pheochromocytoma PC-12 cells and its level increases after differentiation of the cells into sympathetic neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP. This mRNA level does not increase in response to 12-O-tetradecanoylphorbol-13-acetate incapable of inducing differentiation. These results suggest that the smg-25A gene is specifically expressed in nerve tissues and that smg p25A plays a role in some neuronal functions.     

Kinetic analysis of the binding of guanine nucleotide to bovine brain smg p25A

Bovine brain smg p25A, a guanine nucleotide-binding protein with a Mr of about 25,000, bound specifically GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and GDP. The initial velocities of the binding of GTP gamma S to GDP-bound smg p25A and the dissociation of GDP from this protein increased by decreasing Mg2+ concentrations or increasing NaCl concentrations. The initial velocity of the binding of GTP gamma S to GDP-free smg p25A was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from smg p25A limits the binding of GTP to this protein, and suggest that there is a protein stimulating the dissociation of GDP from smg p25A and thereby stimulating the binding of GTP to this protein in mammalian tissues. In fact, the protein stimulating the dissociation of GDP, but not of GTP gamma S, from smg p25A was detected in bovine brain cytosol.     

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.     

Detection of point mutation in the tyrosinase gene of a Japanese albino patient by a direct sequencing of amplified DNA

Summary Enzymatic DNA amplification and direct DNA sequencing were used to detect a mutation in the tyrosinase gene of an albino patient. Single-base change could be detected by direct sequencing. This base change (G to A) is thought to result in an amino acid change (Arg to Gln) in tyrosinase of the patient.     

Prediction of the packing arrangement of strands in β-sheets of globular proteins

A method is proposed for predicting the adjacency order in which strands pack in a -sheet in a protein, on the basis of its amino acid sequence alone. The method is based on the construction of a predicted contact map for the protein, in which the probability that various residue pairs are close to each other is computed from statistically determined average distances of residue pairs in globular proteins of known structure. Compact regions, i.e., portions of the sequence with many interresidue contacts, are determined on the map by using an objective search procedure. The proximity of strands in a -sheet is predicted from the density of contacts in compact regions associated with each pair of strands. The most probable -sheet structures are those with the highest density of contacts. The method has been tested by computing the probable strand arrangements in a five-strand -sheet in five proteins or protein domains, containing 62–138 residues. Of the theoretically possible 60 strand arrangements, the method selects two to eight arrangements as most probable; i.e., it leads to a large reduction in the number of possibilities. The native strand arrangement is among those predicted for three of the five proteins. For the other two, it would be included in the prediction by a slight relaxation of the cutoff criteria used to analyze the density of contacts.     

Breeding ofl-threonine hyper-producer ofEscherichia coli W

Summary l-Threonine hyper-producing mutants were obtained fromEscherichia coli W strain KY-8366, by reducingl-threonine degradation activity and enhancingl-threonine biosynthetic activity. Anl-threonine degradation reaction test using resting cells of KY-8366 suggested that the main pathway ofl-threonine degradation by KY-8366 is via glycine. A strain with reducedl-threonine degradation activity was obtained among those mutants that could not utilizel-threonine as sole nitrogen source. Rifampicin-resistant mutants andl-lysine plus methionine-insentitive mutants were isolated. These mutants showed enhanced aspartokinase levels and accumulated morel-threonine than the parental strains. Mutant H-4290 accumulated 58 g/l ofl-threonine.     

l-threonine production by l-aspartate- and l-homoserine-resistant mutant of Escherichia coli

Summary Growth and l-threonine productivity of l-threonine producer Escherichia coli H-4290 were inhibited by precursor amino acids, l-homoserine and l-aspartate. l-Threonine hyper-producers were isolated among the mutants resistant to l-homoserine and l-aspartate. Mutants H-4351 (Hom^r) and H-4578 (Hom^r, Asp^r) accumulated 22.2 g/l and 24.3 g/l of l-threonine in test tube cultures, while the parental strain H-4290 accumulated 18.2 g/l. The enzyme level of aspartokinase I (first enzyme of the threonine operon) was enhanced 2.3 times (H-4351) and 3 times (H-4578) that of H-4290. Mutant H-4578 accumulated 76 g/l of l-threonine in a 2-l jar fermentor after 75 h cultivation.Abbreviations DAP diaminopimeric acid - Met ^l poor growth in methionine-free medium - AHV -amino--hydroxyvaleric acid - Thr-N^- lack of ability to utilize l-threonine as a nitrogen source - Rif rifampicin - Lys+Met^r resistant to l-lysine and dl-methionine     

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.     

Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA.

T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.