Molecular cloning of the cDNAs coding for the two subunits of soluble guanylyl cyclase from human brain

128

The ␣1-and ␤1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells͞baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3- (

mentioning

confidence: 99%

Human recombinant soluble guanylyl cyclase: Expression, purification, and regulation

Lee¹,

Martin²,

Murad³

2000

128

“…At least two mammalian isoforms for each subunit (␣ 1 ͞␣ 2 and ␤ 1 ͞␤ 2 ) have been identified and are expressed in a tissue-specific manner (16). Both isoforms for each subunit have been cloned and sequenced in human, rat, and bovine (17)(18)(19)(20)(21). The ␣ 1 ͞␤ 1 sGC heterodimer appears to be the most abundantly expressed and widely distributed sGC holoenzyme.…”

mentioning

confidence: 99%

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

Krumenacker¹,

Hyder²,

Murad³

2001

Previous reports that investigated the regulation of the NO͞ soluble guanylyl cyclase (sGC)͞cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17␤-estradiol (E2) regulates the ␣1 and ␤1 subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC ␣1 10% and sGC ␤1 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO͞sGC͞cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.nitric oxide ͉ gene regulation ͉ steroid hormone T he steroid hormone 17␤-estradiol (E2) exhibits dramatic effects in the uterus with respect to hyperemia, gene expression, and proliferation of the endometrium. It is clear that there is a wide array of changes in the expression of multiple genes and biochemical processes within the uterus after E2 exposure. However, as a whole, it is unclear how these responses mediate the hyperemic and subsequent proliferative response of the uterine tissue.To understand how these processes are regulated is essential, because normal physiological, pathological, and pharmacological estrogenic responses are common. For instance, the levels of estrogen are tightly regulated and mediate uterine quiescence and initiation of labor during pregnancy (1). In addition, estrogen levels fluctuate during the estrous cycle of mammals and the menstrual cycle of primates, which mediate the endometrial growth cycle (2). Postmenopausal women frequently are administered estrogen supplements in hormone replacement therapy regimens to reduce the loss of bone mass associated with menopause. Furthermore, use of the anti-estrogen, tamoxifen, for treating breast cancer results in paradoxical estrogenic effects in the uterus (3). Finally, both men and women are exposed to low levels of phyto-estrogens on a daily basis, dependent on dietary and environmental intake (4).Reports indicate that estrogens regulate the NO͞soluble guanylyl cyclase (sGC)͞cGMP cell signaling pathway and the levels of NO and the second messenger, cGMP, in many tissues, including the uterus. Recent and past studies have investigated the levels o...

“…A heme prosthetic group is crucial for the stimulation of the enzyme by NO (10,11). The enzyme has been purified from various animal tissues (10,12,13), and corresponding cDNAs were cloned from various vertebrate species, including rat (14,15), human (16), bovine (17,18), and fish (19). At least two isoforms for each subunit of the enzyme have been identified in various species, prompting a recent revision of the nomenclature of sGC subunits (20).…”

mentioning

confidence: 99%

Genomic organization of α1 and β1 subunits of the mammalian soluble guanylyl cyclase genes

Sharina¹,

Krumenacker

Martin³

et al. 2000

The structures of the genes encoding the ␣1 and ␤1 subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNAs isolated from mouse lungs encoding the ␣1 (2.5 kb) and ␤1 (3.3 kb) subunits are presented in this report. The ␣1 sGC gene is approximately 26.4 kb and contains nine exons, whereas the ␤1 sGC gene spans 22 kb and consists of 14 exons. The positions of exon͞intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome Database predicted the exon͞intron boundaries of the human genes and revealed that human and mouse ␣1 and ␤1 sGC genes have similar structures. Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5 untranscribed regions of ␣1 and ␤1 subunit genes were subcloned into luciferase reporter constructs, and the functional analysis of promoter activity was performed in murine neuroblastoma N1E-115 cells. Our results indicate that the 5 untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, suggest independent regulation of both genes.nitric oxide ͉ chromosomal localization