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M Negishi

Publications and source records attributed to M Negishi.

At least 307 records · Page 17Linked to original sources
PubMed1989

Characterization of a cDNA for rat P-450g, a highly polymorphic, male-specific cytochrome in the P-450IIC subfamily.

Cytochrome P-450g (IIC13) is a highly polymorphic, male-specific rat liver isozyme which is a member of the P-450IIC subfamily. A cDNA, c5126 (1737 bp), for P-450g was isolated from a lambda gt11 library synthesized from (+g) male rat liver mRNA. Sequence analysis of the clone, c5126, revealed an open reading frame of 1473 nucleotides, which encodes for a 490 amino acid polypeptide possessing the 30 NH2-terminal residues reported for cytochrome P-450 (M-3) (P-450g) [Matsumoto et al. (1986) J. Biochem. 100, 1359-1371]. A high degree of sequence similarity (greater than 70%) exists between c5126 and the published sequences of cDNAs for members of the IIC subfamily, while its sequence similarity to other subfamilies (IA, IIB, and IIIA) was much lower (less than 55%). RNA blot analysis utilizing an oligonucleotide probe specific for P-450g revealed that P-450g mRNA was expressed in livers of male but not female Sprague-Dawley (CD) and ACI rats, indicating that the sex difference was regulated pretranslationally. Furthermore, expression of P-450g mRNA was age dependent in livers of male ACI rats (a homozygous, phenotypically high P-450g strain). However, the mRNA for P-450g was expressed equally in livers of outbred male CD rats representing either the high (+g) or the low (-g) phenotype and of inbred ACI rats (+g) representing the high phenotype, indicating that the defect in (-g) rats does not reflect differences in expression of P-450g mRNA.

Amino Acid Sequence↗
PubMed1989

Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue.

As a family of structurally-related enzymes, cytochrome P450 (P450) monooxygenases exhibit paradoxical characteristics: although collectively the enzymes display a broad range of substrate specificities, individually they are characterized by a high degree of substrate and product selectivity. Mouse P45015 alpha and P450coh, for example, which are expressed in female liver and male kidney cells, catalyse 15 alpha-hydroxylation of delta 4 3-ketone steroids, such as testosterone and 7-hydroxylation of coumarin, respectively. In spite of their divergent catalytic activities, however, these enzymes differ by only 11 amino acids within their 494 residues. To determine the structural basis of the different substrate specificities of P45015 alpha and P450coh we therefore altered each of these 11 residues by site-directed mutagenesis, expressing the mutant cytochromes in COS-1 cells. We report that the activities of both cytochromes depend critically on the identities of the amino acids at positions 117, 209 and 365 and, moreover, that a single mutation in which Phe 209 is substituted by Leu is sufficient to convert the specificity of P450coh from coumarin to steroid hydroxylation.

Amino Acid Sequence↗
PubMed1989

Functional characterization of two cytochrome P-450s within the mouse, male-specific steroid 16 alpha-hydroxylase gene family: expression in mammalian cells and chimeric proteins.

Two cDNAs, pc16 alpha-2 and pc16 alpha-25, which encode P-450s from within the mouse, male-specific steroid 16 alpha-hydroxylase (C-P-450(16 alpha)) gene family, were transfected into COS-1 cells in order to study catalytic activities of the expressed P-450s. pc16 alpha-2 was shown previously to encode the growth hormone dependent and androgen-dependent C-P-450(16 alpha) in adult male mice (Wong et al., 1987). The sequence of pc16 alpha-25-encoded P-450 (P-450cb) was identical with gene cb within the C-P-450(16 alpha) family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-450(16 alpha). We expressed both P-450s by transfecting their cDNAs into COS-1 cells and found that steroid 16 alpha-hydroxylase activity was catalyzed by C-P-450(16 alpha) but not by P-450cb. In addition to testosterone, progesterone and estradiol were hydroxylated specifically at the 16 alpha-position by the expressed C-P-450(16 alpha). The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P-450(16 alpha). We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P-450(16 alpha) molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16 alpha-hydroxylase activity.

Amino Acid Sequence↗
PubMed1989

Mouse steroid 15 alpha-hydroxylase gene family: identification of type II P-450(15)alpha as coumarin 7-hydroxylase.

We identified type II P-450(15)alpha as mouse coumarin 7-hydroxylase (P-450coh). Unlike type I P-450(15)alpha, the other member within the mouse steroid 15 alpha-hydroxylase gene family, type II catalyzed little steroid 15 alpha-hydroxylase activity, yet structurally there were only 11 substitutions between type I and type II P-450(15)alphaS within their 494 amino acid residues (Lindberg et al., 1989), and the N-terminal sequence (21 residues) of P-450coh was identical with that of both P-450(15)alphaS. Induction by pyrazole of coumarin 7-hydroxylase activity correlated well with the increase of type II P-450(15)alpha mRNA in 129/J male and female mice. Pyrazole, on the other hand, was less in males or not effective in females in inducing the 15 alpha-hydroxylase activity and type I P-450(15)alpha mRNA. Expression of type I and II in COS-1 cells revealed that the latter catalyzed coumarin 7-hydroxylase activity at 10 to approximately 14 pmol min-1 (mg of cellular protein)-1. The former, on the other hand, had a high testosterone 15 alpha-hydroxylase but little coumarin 7-hydroxylase activity. It was concluded, therefore, that type II P-450(15)alpha is the mouse coumarin 7-hydroxylase. Identification of type II as the P-450 specific to coumarin 7-hydroxylase activity and characterization of its cDNA and gene, therefore, were significant advances toward understanding the basis of genetic regulation of this activity in mice (known as Coh locus).

Animals↗
PubMed1989

The structure and characterization of type I P-450(15) alpha gene as major steroid 15 alpha-hydroxylase and its comparison with type II P-450(15) alpha gene.

The structures of genes 15 alpha OH-1 and 15 alpha OH-2, within the mouse steroid 15 alpha-hydroxylase (P-450(15) alpha) family were determined. Genes 15 alpha OH-1 and -2 encoded mouse Type I and II P-450(15) alpha, respectively (Squires, E.J., and Negishi, M. (1988) J. Biol. Chem. 263, 4166-4171). The two genes, which spanned approximately 8 kilobase pairs of total length, showed nearly identical structures and were divided into nine exons at the same positions. A high nucleotide sequence homology (greater than 96%) indicated that 15 alpha OH-1 and -2 were duplicated within 5 million years. The two major transcription start sites were located at 14 and 24 base pairs (bp) upstream from the initiation Met in both genes. No tissue-specific difference in 15 alpha OH-1 and -2 transcriptional start sites was found in mouse liver and kidney. Both 15 alpha OH-1 and -2 had TATA and CAAT boxes at 30 and 100 bp, respectively, upstream from their major transcription start sites. A glucocorticoid regulatory element was present at 336 bp upstream from the start site in both genes. In addition to these motifs, 15 alpha OH-2 had an SV40 enhancer core sequence immediately downstream from its CAAT box. There were only 11 substitutions between Type I and II P-450(15) alpha in their 494-amino acid residues. Type I cDNA-transfected cell homogenates had testosterone 15 alpha-hydroxylase activity at approximately 9 pmol/min/mg protein. Type I also catalyzed progesterone and androstenedione 15 alpha-hydroxylase activities. Type II, on the other hand, exhibited little activity toward these steroids. The results indicated that Type I was the major steroid 15 alpha-hydroxylase. The differential 15 alpha OH-1 expression, therefore, determined the sexual dimorphism of the tissue-specific 15 alpha-hydroxylase activity in mice.

Amino Acid Sequence↗
PubMed1989

Stimulation of cAMP formation by prostaglandin D2 in primary cultures of bovine adrenal medullary cells: identification of the responsive population as fibroblasts.

In primary cultures of bovine adrenal medulla, chromaffin cells responded to prostaglandin (PG) E2 by stimulating phosphoinositide metabolism (Yokohama et al. (1988) J. Biol. Chem. 263, 1119-1122). In contrast, nonchromaffin cells were found to respond to PGD2 by elevating their intracellular cAMP level. The formation of cAMP was detected at as low as 0.1 nM PGD2 and increased more than 100-fold over the basal level at 0.1 microM, and the response was specific for PGD2 (greater than PGE1 greater than PGE2 greater than PGF2 alpha = PGI2). The magnitude of cAMP formation and its specificity to PGD2 were retained throughout a 40-day culture period. Based on the inhibitory effect of cis-4-hydroxy-L-proline, an inhibitor of collagen synthesis, on cAMP formation, morphology, and immunoreactivity of cells to anti-collagen type I antiserum, the responsive cells were identified as fibroblasts. These results taken together demonstrate that the adrenal medulla is composed of chromaffin and nonchromaffin cells, which respond to PGE2 and PGD2, respectively, by two different signal transduction pathways. The cAMP formation by PGD2 was also observed in fibroblasts from bovine embryonic trachea among cell lines tested, suggesting that some populations of fibroblasts responsive to PGD2 exist in various tissues and may discriminate the signal from that of PGE1 or PGE2.

Adrenal Medulla↗
PubMed1989

Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner.

Prostaglandin E (PGE) receptor is coupled to a pertussis toxin-insensitive GTP-binding protein in bovine adrenal medulla, but PGE receptor partially purified from bovine adrenal medulla was functionally reconstituted with Gi into phospholipid vesicles (Negishi, M., Ito, S., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1988) J. Biol. Chem. 263, 6893-6900). We demonstrate here that PGE2 inhibited forskolin-induced accumulation of cAMP in cultured bovine chromaffin cells. In plasma membranes prepared from bovine adrenal medulla, PGE2 inhibited forskolin-stimulated adenylate cyclase activity in a GTP-dependent manner. This inhibitory action of PGE2 was abolished by treatment of the membrane with pertussis toxin. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi purified from bovine brain restored the potency of PGE2 to inhibit the adenylate cyclase activity. Inhibition of forskolin-induced cAMP accumulation by PGE2 was also abolished by exposure to the toxin in the cells, indicating that PGE receptors are coupled to Gi. In contrast, PGE2 stimulated the formation of inositol phosphates in chromaffin cells, but this effect was not affected by treatment of the cells with pertussis toxin, suggesting that the PGE receptors are coupled to phosphoinositide metabolism via a pertussis toxin-insensitive G-protein. Both the inhibitory action of cAMP accumulation and stimulation of phosphoinositide metabolism were specific for PGE1 and PGE2, and the Scatchard plot analysis of PGE2 binding to the membrane showed a single high-affinity binding site (Kd = 2 nM). In bovine adrenal chromaffin cells PGE2 enhanced catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). We further examined the modulation of catecholamine release by PGE2 through its inhibitory coupling to the adenylate cyclase system. Prior exposure of chromaffin cells to forskolin or dibutyryl-cAMP reduced nicotine-stimulated catecholamine release, and PGE2 attenuated forskolin-induced inhibition of catecholamine release stimulated by nicotine, but not dibutyryl-cAMP-induced inhibition. In the absence of evidence that PGE receptor subtypes exist, these results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.

Adenosine Diphosphate Ribose↗
PubMed1989

Gene family of male-specific testosterone 16 alpha-hydroxylase (C-P-450(16 alpha)) in mice. Organization, differential regulation, and chromosome localization.

Three genes in the testosterone 16 alpha-hydroxylase (C-P-450(16 alpha)) family, ca, cb, and cc, are characterized. The sizes of the genes are approximately 4.5 to 5.2 kilobase pairs, and all three consist of nine exons with junctions at identical sites. Gene ca is identified as the male-specific, androgen-dependent C-P-450(16 alpha) gene in adult mice, since the exonic sequence matched 100% to the cDNA, pc16 alpha-2 (Wong, G., Kawajiri, K., and Negishi, M. (1987) Biochemistry 26, 8683-8690). Gene cb and cc are organized in tandem within 18-kilobase pair DNA. Their encoded P-450s contain an approximate 94% nucleotide sequence similarity to the C-P-450(16 alpha). The high similarity in gene nucleotide sequences, including the introns and flanking regions, suggests a combination of an ancestral gene duplication and gene conversion as a mechanism for evolution of the C-P-450(16 alpha) family. Gene ca shows male-specific expression in mouse kidney as well as in liver; gene cb, neither sex-specific nor androgen-dependent, is seen only in liver; gene cc is not expressed in either adult mouse liver or kidney. Expression of these three genes is not detected in adult mouse lung. It appears, therefore, that the C-P-450(16 alpha) gene family includes a large number of genes whose expressions are differentially regulated. Southern hybridization of C-P-450(16 alpha) cDNA to genomic DNAs from mouse-hamster somatic hybrid cells localizes tentatively this gene family on mouse chromosome 15. The recombination frequency in BXD recombinant inbred mice suggests that the C-P-450(16 alpha) gene family is approximately 16M from the 55-kDa locus.

Animals↗
PubMed1989

Detection of high levels of immunoreactive human beta-1 interferon in sera from HIV-infected patients.

We have examined human interferon beta-1 (HuIFN-beta 1) levels in the sera from patients with various diseases by using a newly developed highly sensitive enzyme-linked immunosorbent assay system (ELISA). High levels of HuIFN-beta 1 were detected in the sera from Human Immunodeficiency Virus-I (HIV-I) infected patients, especially of asymptomatic carriers. The serum level of HuIFN-beta 1 may be a good indicator of HIV-I infection and of other immunological disorders.

AIDS-Related Complex↗
PubMed1989

Dynorphin A (1-13), microinjected into the preoptic area, stimulates water intake in rats.

The effects of dynorphin A (1-13) (DYN), injected into the preoptic area, was investigated on water intake in rats. DYN at both doses of 2 and 10 nmoles significantly increased water intake for two and four hours after the injection in a dose related fashion. However, no significant change was observed in food intake. Naloxone pretreatment (0.3 mg/kg, s.c.) completely attenuated the DYN-induced stimulation of water intake. The present studies suggest that DYN in the preoptic area may play an important role in the regulation of drinking behavior, but have no effect on food intake.

Animals↗
PubMed1989

Production of interleukin 1-like factor from human peripheral blood monocytes and polymorphonuclear leukocytes by superoxide anion: the role of interleukin 1 and reactive oxygen species in inflamed sites.

In the present study, we investigated the possibility that oxidative stress to human peripheral blood monocytes and polymorphonuclear leukocytes (PMNs) can induce interleukin 1 (IL-1)-like activity. Oxidative stress that we used was superoxide anion (O2-) and hydrogen peroxide (H2O2). O2-, but not H2O2, could induce an IL-1-like factor(s) from monocytes and PMNs. IL-1-like activity from monocytes and PMNs induced by O2- was due to de novo synthesis because no IL-1-like activity was found in culture supernatants and in the lysate of unstimulated cells. We next examined the effects of radical scavengers on production of an IL-1-like factor(s). Generation of IL-1-like activity from monocytes was amplified by preincubation with catalase (H2O2 scavenger), although it was suppressed by preincubation with either superoxide dismutase (O2- scavenger) or vitamin E (antioxidant analogs). These results suggest that production of an IL-1-like factor(s) from monocytes and PMNs was due to O2- stimulation. Our data that production of an IL-1-like factor(s) from inflammatory cells by stimulation with O2- imply a model of the up-regulation mechanism of inflammation mediated by enhanced IL-1-like factor production stimulated with reactive oxygen species.

Glucose Oxidase↗
PubMed1989

[Clinical trial of T-3262 on acute enteritis. Japan Research Committee of T-3262, Research Group for Acute Infectious Enteritis].

For the purpose of evaluation of clinical efficacy, safety and usefulness on acute infectious enteritis (bacillary dysentery, and enteritis caused by Salmonella spp., Campylobacter spp., enteropathogenic E. coli, and so on), T-3262, a newly developed pyridone-carboxylic acid derivative, was administered to a total of 136 patients and carriers. In addition, in vitro antibacterial activity of T-3262 was determined against the clinical isolates, and compared with those of nalidixic acid (NA), pipemidic acid (PPA), enoxacin (ENX), norfloxacin (NFLX) and ofloxacin (OFLX). The daily dose of 450 mg of T-3262 was administered orally three times after meals for 5 days, with the exception of 7 day administration against Salmonella enteritis. A total of 89 cases were evaluated; 23 with Shigella spp., 30 with Salmonella spp., 15 with Campylobacter spp., 6 with enteropathogenic E. coli, and 15 cases with the other pathogens or pathogen-negative. The efficacy on clinical symptoms judging from duration of fever, and duration of diarrhea and abnormal stool character was 100% in all the enteritis except enteropathogenic E. coli enteritis, in which it was 50% (n = 2). Concerning bacteriological response, elimination of the causative organisms from the feces was 100% in Shigella spp. (n = 19), Salmonella spp. (n = 30), and enteropathogenic E. coli (n = 6), although 64.3% in Campylobacter spp. (n = 14). As an adverse effect, epigastric discomfort was observed in one (0.8%) of 130 cases. Deteriorations in laboratory findings were seen in five (6.2%) of 81 cases, consisting of two with elevated GOT and GPT, two with elevated GPT, and one with increased eosinophils count, although they were all slight in degree. MICs of T-3262 which inhibited 90% of the isolates of Shigella spp, Salmonella spp., and Campylobacter spp., were 0.025, 0.05, and 0.78 microgram/ml, respectively. These values were lowest among the quinolone derivatives tested, except that the MIC90 against Campylobacter spp. was the same as that of ofloxacin.

4-Quinolones↗
PubMed1988

Inhibition of prostaglandin E2-induced phosphoinositide metabolism by phorbol ester in bovine adrenal chromaffin cells.

In bovine adrenal chromaffin cells, prostaglandin E2 (PGE2) stimulates the formation of inositol phosphates and Ca2+ mobilization through its specific receptor [Yokohama, Tanaka, Ito, Negishi, Hayashi & Hayaishi (1988) J. Biol. Chem. 263, 1119-1122]. Here we show that PGE2-induced phosphoinositide metabolism was blocked by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using intact cells, we also examined the inhibitory effect of TPA on the individual steps of the activation process of phosphoinositide metabolism. The inhibition was observed within 1 min and complete by 10 min after addition of 1 microM-TPA, and half-maximal inhibition by TPA occurred at 20 nM. TPA prevented Ca2+ mobilization induced by PGE2, but not by the Ca2+ ionophore ionomycin. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not inhibit the formation of inositol phosphates and Ca2+ mobilization by PGE2. TPA treatment affected neither the high-affinity binding of [3H]PGE2 to intact cells and membrane fractions nor the ability of guanosine 5'-[gamma-thio]triphosphate to decrease the binding in membrane fractions. TPA also abolished phosphoinositide metabolism induced by muscarinic-receptor activation. NaF plus AlCl3 and ionomycin caused the accumulation of inositol phosphates, probably by directly activating a GTP-binding protein(s) and phospholipase C respectively; neither accumulation was inhibited by TPA treatment. These results suggest that protein kinase C serves as a feedback regulator for PGE2-induced phosphoinositide metabolism. The site of action of TPA appears to be distal to the coupling of the receptor to GTP-binding protein, but on a component(s) specific to the agonist-induced phosphoinositide metabolism.

Adrenal Medulla↗
PubMed1988

Rip locus: regulation of female-specific isozyme (I-P-450(16 alpha) of testosterone 16 alpha-hydroxylase in mouse liver, chromosome localization, and cloning of P-450 cDNA.

The constitutive expression of phenobarbital-inducible mouse cytochrome P-450 (I-P-450(16 alpha) at the mRNA level and its associated testosterone 16 alpha-hydroxylase activity in liver microsomes was a female characteristic in many inbred mice, including BALB/cJ, A/HeJ, and C57BL/6J. This sex-dependent constitutive expression of the mRNA and enzyme activity was severely reduced in females of mouse strain 129/J. The distribution patterns of the mRNA and activity levels in individual offspring of F1, F2, and F1 backcrosses to progenitors, generated from crosses between 129/J and BALB/cJ mice, indicated that the female-specific expression of I-P-450(16 alpha) is an autosomal dominant trait under the regulation of a sex-limited single locus. It was found that the genotypes of this locus exhibited concordance with that of the coumarin hydroxylase locus (Coh locus) in eight out of nine 9 X A recombinant inbred strains, suggesting the localization of this sex-limited locus on chromosome 7. We propose Rip (regulation of sex-dependent, constitutive expression of phenobarbital-inducible P-450) as the name of this sex-limited locus. With the use of the rat P-450e cDNA probe, a cDNA library from liver poly(A+) RNA of BALB/cJ was screened, and three distinct cDNAs (pf3, pf26, and pf46) were selected on the basis of their restriction patterns. Nucleotide sequences of the cDNAs revealed that pf3 and pf46 are clones overlapped, with the exception that the 27-bp DNA is inserted in the coding region of pf46. The nucleotide sequence (named pf3/46) obtained from the overlapping sequences of pf3 and pf46 contained 1473 or 1500 bp of open-reading frame, and the deduced amino acid sequence shared 93% similarity with those of rat P-450b. The 27-bp insertion resulted in nine extra amino acids just in front of the cysteine residue, the fifth ligand for heme binding. The mRNA with 27-bp insertion was ubiquitously present in other inbred mice such as A/HeJ and C57BL/6J, but not in 129/J. S-1 nuclease analysis estimated a ratio of p46 and pf3 to be 1:50. Nucleotide and deduced amino acid sequences of the 1473-bp open-reading frame in pf26 possessed 83% similarity to those of pf3/46. Hybridizations of oligonucleotide probes (pf26-cu and pf3/46-cu) specific to either pf26 or pf3/46 with liver poly(A+) RNA from males and females of BALB/cJ, 129/F, and F1 offspring demonstrated that the expression of pf26, but not pf3/46, mRNA was associated with the autosomal dominant inheritance of I-P-450(16 alpha).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1988

Genetic regulation of estrogen-dependent repression of female-specific testosterone 16 alpha-hydroxylase (I-P-450(16 alpha) in male mouse liver: murine Ripr locus.

The genetic basis for repression of I-P-450(16 alpha) in livers of male mice was examined in 129/J and BALB/cJ mice. Castration of adult male BALB/cJ but not 129/J mice resulted in derepression of I-P-450(16 alpha) at its mRNA and activity levels. It was further found that the patterns of derepression in (129/J x BALB/cJ) F1 and F2 offspring indicated that the derepression of I-P-450(16 alpha) is inherited as an autosomal additive trait. The distribution of derepression among castrated recombinant inbred strains (9 X A) indicated a close link of a locus repressing I-P-450(16 alpha) in male mice to the Rip locus on chromosome 7. Rip was previously defined as a locus that regulates specific expression of I-P-450(16 alpha) in livers of female mice [Noshiro, M., Lakso, M., Kawajiri, K., & Negishi, M. (1988) Biochemistry (preceding paper in this issue)]. Other tested inbred mice (A/HeJ, C57BL/6J, C3H/HeJ, and DBA/2J) showed the derepression of I-P-450(16 alpha) by castration, such as BALB/cJ. We propose Ripr (repression of an action of Rip locus in male mice) as the name of the locus by which repression of I-P-450(16 alpha) is regulated in male mice. Treatment of castrated male BALB/cJ mice by testosterone propionate, estradiol valerate, or diethylstilbestrol repressed I-P-450(16 alpha) to the levels seen in normal BALB/cJ male mice. Dihydrotestosterone, however, had little effect in repressing I-P-450(16 alpha) in castrated mice. The results suggested that estrogen rather than androgen is a repressor of I-P-450(16 alpha) in livers of male mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins.

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.

Adrenal Medulla↗