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.     

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.     

Characterization of antisera to 2-hydroxyestradiol and 4-hydroxyestradiol using 6-(O-carboxymethyl)oxime- and 17-hemisuccinate-bovine serum albumin conjugates and radioimmunoassay

For radioimmunoassay of the catechol estrogens, four hapten-bovine serum albumin (BSA) conjugates were prepared from 6-oxo-2-hydroxyestradiol 6-(O-carboxymethyl)oxime, 2-hydroxyestradiol 17-hemisuccinate, 6-oxo-4-hydroxyestradiol 6-(O-carboxymethyl)oxime and 4-hydroxyestradiol 17-hemisuccinate by coupling with BSA, employing the mixed anhydride method. The antisera elicited in rabbits by immunization with these antigens showed high affinity and specificity for 2-hydroxyestradiol or 4-hydroxyestradiol with cross-reactivities to a few structurally related estrogens. The specificity of antisera obtained is discussed in relation to the site of attachment of the hapten to BSA.     

Phorbol ester increases the level of interleukin 2 mRNA in mitogen-stimulated human lymphocytes

TPA alone did not induce the production of IL 2 in human tonsillar lymphocytes but enhanced the PHA-induced IL 2 production by seven-fold. That the effect of TPA was due to an increase in IL 2 mRNA was demonstrated by examining the amount of IL 2 mRNA translatable in Xenopus laevis oocytes, and by Northern blotting analysis using IL 2 cDNA as a probe. In these ways, it was shown that TPA alone did not induce any significant IL 2 mRNA synthesis, but when added together with PHA it increased the level of IL 2 mRNA by at least 10-fold, as compared with that induced by PHA alone.     

LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1.

The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).     

Translation of human interleukin 2 mRNA in Xenopus laevis oocytes

Poly(A)-positive mRNA extracted from tonsillar mononuclear cells stimulated with phytohemagglutinin-M and 12-o-tetradecanoyl phorbol 13-acetate was successfully translated into biologically active interleukin 2 (IL-2) in Xenopus laevis oocytes, and secreted into the incubation medium. In control experiments, the extract of oocytes injected with either poly(A)-negative RNA or buffer did not show any IL-2 activity. By sucrose density gradient centrifugation analysis, IL-2 mRNA was found as a single peak corresponding to a sedimentation coefficient of 10-11S.     

Peroxisomal acetoacetyl-CoA thiolase of an n-alkane-utilizing yeast, Candida tropicalis.

Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambda EMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41,898 Da for thiolase IA and 41,930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto.