Masahide Kasugai

Purification and properties of human placental aminopeptidase B

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.

Purification and properties of microsomal carboxypeptidase N (kininase I) in human placenta.

Carboxypeptidase N (kininase I, EC 3.4.17.3) was found in human placenta and purified 600-fold. The enzyme was solubilized from membrane fractions with Triton X-100 and was purified by affinity chromatography with histargin, a potent inhibitor of this enzyme. The pH optimum of the enzyme was 7.8. The Km values for L-hippuryl-L-lysine and bradykinin were 1.25 and 0.43 mmol/l, respectively. The apparent molecular mass (Mr) of the enzyme determined by gel filtration was estimated to be 280,000, which is identical to that of the human serum enzyme. We propose that the placenta is a major source of carboxypeptidase N and thus may be involved in the physiological control of fetal circulation by regulating the kallikrein-kinin and renin-angiotensin systems.

Rapid determination of fetal sex using amniotic fluid cells and the polymerase chain reaction

SummaryWe determined the sex of 50 fetuses by an amplification of the Y-chromosome specific fragment using the polymerase chain reaction (PCR). Amniotic fluid cells were collected by amniocentesis from pregnant women at 14 to 17 weeks of gestation. Total DNA was purified from cells in 1 ml of amniotic fluid. When only the expected 130 base pair X-chromosome specific fragment was detected, we identified the fetus as female, while when both the expected 170 base pair Y-chromosome specific and X-chromosome specific fragments were detected, we identified it as male. In all cases, identification was confirmed either by chromosome analysis or post partum.

Enhanced proliferation of fetal rat hepatocytes in primary culture induced by ritodrine

OBJECTIVE Although ritodrine crosses the placenta, its direct effect on fetal cell proliferation has not been reported. We hypothesized that beta 2-adrenergic receptor stimulation could promote fetal liver growth. STUDY DESIGN Ritodrine was added to serum- and hormone-free primary cultures of fetal, neonatal, or adult rat hepatocytes. We measured both tritiated thymidine incorporation into deoxyribonucleic acid and nucleus number. The effect of ritodrine on cell cycle was also analyzed with flow cytometry. RESULTS Ritodrine enhanced the proliferation of fetal rat hepatocytes. Ritodrine remarkably stimulated deoxyribonucleic acid synthesis of fetal and neonatal but not adult hepatocytes. The effect was dose dependent and was antagonized by propranolol. Analysis of the nuclear deoxyribonucleic acid content derived from flow cytometry revealed that cells stimulated by ritodrine entered S phase. CONCLUSION These results indicate that ritodrine may promote the proliferation of fetal hepatocytes through the stimulation of beta 2-adrenergic receptors, followed by induction of deoxyribonucleic acid synthesis.