Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C 1 and C 2 ). The first cytoplasmic domain of type I enzyme (IC 1 ) and the second cytoplasmic domain of type II enzyme (IIC 2 -⌬3, a construct in which 36 N-terminal amino acids of the C 2 region are deleted) were expressed and purified to homogeneity. Alone, each had no adenylyl cyclase activity; however, mixing of the two domains in vitro resulted in G s␣ -and forskolin-activated enzyme activity. The turnover number for G s␣ -and forskolin-stimulated enzyme activity of the complex between IC 1 and IIC 2 -⌬3 was 8.2 s ؊1 . The concentration of IIC 2 -⌬3 to achieve half-maximal activation of IC 1 was 0.8 and 1.3 M when stimulated by forskolin and G s␣ , respectively. The concentration of IIC 2 -⌬3 needed to complex with IC 1 was reduced 10-fold (0.08 M) when the enzyme was activated by both forskolin and G s␣ , suggesting that G s␣ and forskolin increased the affinity of the two cytoplasmic domains for each other.The enzymatic activity of adenylyl cyclase is the key step in regulating the intracellular cAMP concentration upon stimulation of a variety of hormones, neurotransmitters, and other regulatory molecules. There are at least nine distinct mammalian adenylyl cyclases which have a similar structure (Fig. 1A) (1-11). This includes two intensely hydrophobic domains (M 1 and M 2 ) and two ϳ40-kDa cytoplasmic domains (C 1 and C 2 ). The C 1 and C 2 domains contain sequences (C 1a and C 2a ) that are similar to each other and to other adenylyl and guanylyl cyclases (12, 13). Each isoform of adenylyl cyclase has its own distinct tissue distribution and unique regulatory properties, providing modes for different cells to respond diversely to similar stimuli (12,14).Membrane-bound adenylyl cyclases are expressed in small quantities, and the enzyme is labile and difficult to manipulate in detergent-containing solutions. To facilitate biochemical and structural analysis, a soluble adenylyl cyclase has been constructed by linking the C 1a and C 2a domains of type I and type II adenylyl cyclases, respectively (15). The resulting protein is sensitive to activation by G s␣ 1 and forskolin and to inhibition by P-site inhibitors, indicating the essential roles of C 1a and C 2a domains for catalysis and regulation. In this paper, we describe the expression and purification of the C 1a and C 2a domains of type I and type II adenylyl cyclase, respectively. Alone, each has no adenylyl cyclase activity; however, mixing of the two domains in vitro results in G s␣ -and forskolin-activated enzyme activity. EXPERIMENTAL PROCEDURESPlasmids-For construction of the expression plasmid vector pProEx-HAH6, the NcoI and EcoRI 4.9-kb fragment of pProEx-1 (Life Technologies, Inc.) was ligated with the phosphorylated linkers (5Ј-CATGCATCACCATCACCATCACGCGGCCGCCTACCCGTATGATGT-CCCGGATTACGCCGGAATTCCCATGGC and 5Ј-AATTGCCATGGGA-ATTCCGGCGTAATCCGGGACATCATACGGGTAGGCGGCCGCGTG-TGGTGATGGTGATG). Proper insertion of cDNA at the NcoI site of pProEx-HAH6 vector would result in the expres...
After completion of this article, the reader will be able to Compare different surgical treatments for pelvic congestion syndromes associated with pelvic pain syndromes. Estimate the relative severity of pelvic congestion in women using current venographic criteria. Choose between different diagnostic methods for characterizing pelvic venous blood flow and anatomy in women presenting with pelvic pain.
Radiolabeled putrescine is a potential imaging agent for carcinoma of the prostate. The intrinsically high uptake of putrescine into the rat prostate and prostatic carcinoma can be further stimulated by pretreatment of the animals with alpha-difluoromethylornithine (DFMO) uptake, a polyamine synthesis inhibitor. Pharmacological castration of the animals followed by androgen stimulation and DFMO pretreatment achieved a 30:1 prostate/muscle ratio of 14C-putrescine uptake in elderly rats.
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