Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.

Abstract

The non-stimulated (basal) adenylate cyclase from bovine brain cortical membranes was purified 10 000-fold to apparent homogeneity by Lubrol PX extraction and two cycles of affinity chromatography on forskolin-agarose. The final product appears as one major band (mol. wt. 115 000) on SDS-polyacrylamide gels. Further identification was achieved by affinity cross-linking using Gs (stimulatory GTPbinding protein) that was [32P]ADP-ribosylated by choleratoxin/[32P]NAD: cross-linking with disuccinimidyl suberate gave products with mol. wts. of 160 000, -270 000 and higher. The distribution of these products was dependent on the concentration of cross-linker, suggesting aggregation of two or more adenylate cyclase complexes. In contrast, photoaffinity cross-linking with 4-azidobenzoyl-[32P]Gs yielded a single product with a mol. wt. of 160 000. Purified adenylate cyclase was completely unresponsive towards stimulators (GTP-analogs, NaF) acting via Gs suggesting that this component was removed during purification. On the other hand, stimulation by forskolin and by added activated Gs was preserved but to a smaller degree as compared with the crude enzyme. In contrast, the stimulation of Ca2+/calmodulin was only marginal. Purified adenylate cyclase reversibly bound to wheat germ agglutinin-Sepharose. This suggests that bovine brain adenylate cyclase is a glycoprotein.

Mechanism of Isoprenaline-Stimulated Diacylglycerol Formation in Rat Parotid Acinar Cells

“…The details of the purification will be described elsewhere. The specific activity of the purified enzyme (5 6 ,umol cyclic AMP formed/min/mg protein in the presence of 100 pM forskolin) was comparable to that reported by Pfeuffer et al (9). The purified enzyme preparation contained proteins of Mr 180,000 and 135,000, whose molecular weights were similar to those of the catalytic subunits (Mr 175,000 and 140,000) reported by Orland et al (8) ( Fig.…”

“…The catalytic subunit of adenylate cyclase used in this study was highly purified from rat brain by chro matography on forskolin-agarose and DEAE-5PW col umns, essentially as described by Pfeuffer et al (9). The details of the purification will be described elsewhere.…”

Regulation of signal transfer from beta1-adrenoceptor to adenylate cyclase by betagamma subunits in a reconstituted system

“…Adenylate cyclase was purified from bovine brain as described by Pfeuffer et a]. [21]. The purity of G, protein, neutrophil a subunit and the by subunits is documented in Fig.…”

“…In the experiment in Fig. 8, lipid vesicles were prepared containing pure turkey erythrocyte PIreceptor, G, and a 2 -8-molar excess of pure turkey erythrocyte Py subunits and the purified catalytic subunit of adenylate cyclase from bovine brain [21]. In this reconstituted R .…”

“…8. These experiments were carried out with pure turkey erythrocyte membrane P1-adrenoceptor, pure G, preparations and pure bovine brain adenylate cyclase [21]. Again, addition of bovine brain by subunits effectively inhibited cyclase activity, whereas ROS transducin By subunits were ineffective up to 200 nM concentrations.…”