Use of a Competitive Protein-Binding Assay for Adenosine 3′:5′-Phosphate for the Study of Bovine Corpus Luteum Adenylate Cyclase

1. Sepharose 6B gel-filtration analysis of soluble adenylate cyclase from bovine corpus luteum is described. Both zonal and frontal techniques of analysis were used. 2. Under conditions of zonal analysis recoveries of activity were low. It was concluded that dissociation of two or more components of the adenylate cyclase complex was occurring on the column and that the maintenance of the complex was essential for the high-activity state of the catalytic unit. Two peaks of adenylate cyclase activity, of approximate mol. wts. 45,000 and 160,000 were detected. 3. The theory of frontal analysis (or steady-state gel filtration), applied to the study of the interacting components of the adenylate cyclase complex is discussed, and activity profiles are predicted. Activity profiles obtained experimentally be frontal analysis compared well with the theoretically predicted profile and provide evidence that dissociation of a high-activity complex, with concomitant loss of activity, does occur. Recoveries of activity under conditions of frontal analysis were higher than with zonal analysis. 4. The effects of concentration and removal of detergent on the activity of the soluble enzyme are discussed.

Section: Methodsmentioning

confidence: 99%

Gel-filtration analysis of soluble adenylate cyclase from bovine corpus luteum

Young¹,

Stansfield²

1978

“…The washed 600g (ray. = 23cm) sediment of bovine corpus-luteum homogenate was prepared as described previously (Young & Stansfield, 1977 Adenylate cyclase activity of the 600g sediment was solubilized in either Lubrol-PX (lOg/I), Triton X-100 (lOg/l) or digitonin (lOg/I) in 40mm-Tris/HCI buffer (pH7.5) containing either 6mM-MgSO4 or 1 mM-MgSO4/1 mM-EDTA, and either with or without NaF (see the Results and Discussion section and legends for variable additions). The washed 600g sediment was resuspended in the detergent solution at a concentration equivalent to 75mg wet wt.…”

Section: Methodsmentioning

confidence: 99%

“…Adenylate cyclase activity was assayed, as described previously (Young & Stansfield, 1977, 1978, in the presence of 40mM-Tris/HCI (pH7.5), 6mM-MgSO4, 1 mM-ATP and 6.7M-caffeine (see legends for variable additions). The total assay volume of 1 ml contained 600g sediment, or soluble extract thereof, equivalent to 37.5mg wet wt.…”

Section: Methodsmentioning

confidence: 99%

Solubilization of bovine corpus-luteum adenylate cyclase in lubrol-PX, triton X-100 or digitonin and the stabilizing effect of sodium fluoride present in the solubilization medium

Young¹,

Stansfield²

1978

1. Adenylate cyclase activity of the washed 600g sediment of bovine corpus-luteum homogenate was solubilized by Lubrol-PX, Triton X-100 and digitonin. Digitonin was the least destructive of NaF-stimulated activity. 2. NaF, present in the solubilization medium together with MgSO4, increased the percentage yields of soluble activity from untreated 600g sediment and 600g sediment which had been preincubated with p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate). The stabilizing influence of NaF was most marked with digitonin. However, the highest specific activities of soluble enzyme were obtained with Lubrol-PX as solubilizing agent, since digitonin solubilized more membrane protein than does Lubrol-PX, and less of the activity of the digitonin-dispersed 600g sediment was recovered in the 105000g supernatant. 3. p[NH]ppG also has a stabilizing effect when present during the solubilization, but less so than NaF. 4. Both NaF and MgSO4 alone have a stabilizing effect during solubilization. The greatest amounts of soluble activity were obtained with both agents present in the solubilization medium, there being a synergistic effect.

“…Cyclic AMP was determined in the boiled supernatant by a competitive protein-binding assay (Young & Stansfield, 1977). All assays were performed in duplicate, and results shown are the mean values with the range of duplicates indicated.…”

Section: Preincubation Conditionsmentioning

confidence: 99%

Activation of adenylate cyclase in bovine corpus-luteum membranes by human choriogonadotropin, guanine nucleotides and NaF

Lydon

Young

Stansfield

1981

1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a ;burst' phase of cyclic AMP synthesis when GTP was present in the assay incubation and a ;lag' phase with p[NH]ppG (guanosine 5'-[beta,gamma-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.