The relationship between the assay values of the activities of the stimulatory
guanine nucleotide-binding regulatory component (N-
protein) of
adenylate cyclase by two different methods: reconstitution of plasma membranes of cyc- S49 cells and ADP ribosylation catalyzed by
cholera toxin, have not been fully elucidated yet. In the present study, the reconstitution and ADP ribosylation assay methods were utilized, and the relationship between the assay values of the N-
protein activities of the erythrocyte membranes from normal subjects and patients with
pseudohypoparathyroidism type I (PHP-I) measured by the two methods was investigated. When
Lubrol extracts of human erythrocyte membranes were reconstituted with cyc- membranes, the rate of
cyclic AMP synthesis reached a constant rate after incubation of 80 minutes at 30 degrees C. This reaction depended on the concentrations of cyc- membranes and
Lubrol extracts of the erythrocyte membranes. N-
protein activity in the erythrocyte membranes of normal subjects and PHP-I patients assayed by reconstitution of cyc- membranes (mean +/- SD, expressed by % of pooled standard preparation) was 100.1 +/- 13.5 (n = 29) and 82.3 +/- 28.0 (n = 19) respectively. The
cholera toxin-catalyzed transfer of [32P]
ADP ribose from [32P]
NAD to the 42,000-dalton
peptide subunit of human erythrocyte N-
protein reached a plateau after incubation of 10 minutes at 30 degrees C. This reaction depended on the concentrations of
cholera toxin,
NAD, and the erythrocyte membranes. N-
protein activity in the erythrocyte membranes from normal subjects and PHP-I patients assayed by
cholera toxin-catalyzed ADP ribosylation (pmol/mg of
protein) was 1.33 +/- 0.20 (n = 7) and 1.15 +/- 0.43 (n = 5) respectively. The correlation between assay values of erythrocyte N-
protein activity in PHP-I patients by reconstitution of cyc- membranes (X, % of pooled standard) and
cholera toxin-catalyzed ADP ribosylation (Y, % of pooled standard) was recognized as a linear regression: Y = 0.77X + 16.8 (r = 0.981, P less than 0.001). It is thus concluded that the activity of N-
protein in human erythrocyte membrane can be assayed by the ADP ribosylation method more simply than by the method of reconstitution of cyc- membranes, with highly close correlation between the values determined by these two methods.