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Journal Article
Research Support, Non-U.S. Gov't
Loss of biological activity of arginine vasopressin during its degradation by vasopressinase from pregnancy serum.
Clinical Endocrinology 1995 January
BACKGROUND AND OBJECTIVE: Degradation of AVP by placental vasopressinase may precipitate gestational diabetes insipidus, which in some cases is accompanied by pre-eclampsia. Abnormally elevated vasopressinase has also been reported in pre-eclampsia without diabetes insipidus. This association between excessive vasopressinase production and pre-eclampsia might be explained if the products of AVP degradation by vasopressinase retained pressor activity even after anti-diuretic activity had been destroyed. Recent evidence indicates that such products may raise blood pressure in rats. The objective of this study was, therefore, to purify vasopressinase and investigate its action on both the V1 and V2 receptor-stimulating activity of AVP.
DESIGN: Vasopressinase was purified from pooled pregnancy serum by ammonium sulphate precipitation, followed by sequential ion exchange, lentil lectin affinity and gel filtration chromatography. Purified enzyme was then used to degrade AVP and the loss of both immunoreactivity and biological activity monitored. Loss of V1 receptor-stimulating activity and V2 receptor-stimulating activity was compared by two-way ANOVA.
PATIENTS: Blood was obtained from healthy women between week 34 and the end of pregnancy. Pooled serum from 20-30 patients was used as starting material for the purification of vasopressinase.
MEASUREMENTS: AVP immunoreactivity was measured by radioimmunoassay, V1 receptor-stimulating activity by a platelet aggregation bioassay, and V2 receptor-stimulating activity by adenylate cyclase stimulation in LLC-PK1 target cells.
RESULTS: Purified vasopressinase was a dimeric protein of molecular weight 330 kDa, which cleaved the synthetic substrate S-benzyl-L-cysteine-4-nitroanilide with a Km of 0.33 mM. Incubation of AVP (0.1 mM) with vasopressinase (0.66 milligrams) at 37 degrees C led to a parallel loss of both AVP immunoreactivity and biological activity. The rates of loss of V1 and V2 receptor mediated activities were not significantly different.
CONCLUSIONS: We report the first direct comparison between the loss of V1 and V2 receptor mediated activities of vasopressinase degraded AVP. There was no significant retention of V1, relative to V2, receptor mediated activity. AVP degradation products are unlikely to be pathogenic in hypertensive pregnancy.
DESIGN: Vasopressinase was purified from pooled pregnancy serum by ammonium sulphate precipitation, followed by sequential ion exchange, lentil lectin affinity and gel filtration chromatography. Purified enzyme was then used to degrade AVP and the loss of both immunoreactivity and biological activity monitored. Loss of V1 receptor-stimulating activity and V2 receptor-stimulating activity was compared by two-way ANOVA.
PATIENTS: Blood was obtained from healthy women between week 34 and the end of pregnancy. Pooled serum from 20-30 patients was used as starting material for the purification of vasopressinase.
MEASUREMENTS: AVP immunoreactivity was measured by radioimmunoassay, V1 receptor-stimulating activity by a platelet aggregation bioassay, and V2 receptor-stimulating activity by adenylate cyclase stimulation in LLC-PK1 target cells.
RESULTS: Purified vasopressinase was a dimeric protein of molecular weight 330 kDa, which cleaved the synthetic substrate S-benzyl-L-cysteine-4-nitroanilide with a Km of 0.33 mM. Incubation of AVP (0.1 mM) with vasopressinase (0.66 milligrams) at 37 degrees C led to a parallel loss of both AVP immunoreactivity and biological activity. The rates of loss of V1 and V2 receptor mediated activities were not significantly different.
CONCLUSIONS: We report the first direct comparison between the loss of V1 and V2 receptor mediated activities of vasopressinase degraded AVP. There was no significant retention of V1, relative to V2, receptor mediated activity. AVP degradation products are unlikely to be pathogenic in hypertensive pregnancy.
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