Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

Effect of α-Methylphenylalanine and Phenylalanine on Brain Polyribosomes and Protein Synthesis

Abstract: A chronic hyperphenylalanemia was effectively produced in developing mice by daily administrations of phenylalanine (2 mg/g body wt) and a phenylalanine hydroxylase inhibitor α-methyl-D, L-phenylalanine (0.43 mg/g body wt). The presence of α-methylphenylalanine in newborn mice inhibited 65–70% of hepatic phenylalanine hydroxylase activity within 12 h. Since this maximum inhibition persisted for 24 h or longer, decreased enzyme activity was maintained by daily administrations. Whereas concentrations of phenylalanine increased approximately 40-fold in both plasma and brain following injection of α-methylphenylalanine and phenylalanine, plasma levels of tyrosine were not altered significantly. Concomitant with changes in phenylalanine concentrations we observed the brain polyribosomes' disaggregation, which reached a maximum 3 h after injection and persisted as long as 18 h. Polyribosomes did not become refractory to as many as 10 daily injections of α-methylphenylalanine and phenylalanine. In addition to polyribosome disaggregation, chronic hyperphenylalanemia reduced the rates of polypeptide chain elongation on polyribosomes isolated from brain homogenates.     

Isolation of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase from Human Brain

Abstract: The enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) has been isolated from an acetone powder of human subcortical white matter. The yield was about 11 mg from 28 g of powder and a specific activity of 213 unitdmg protein was obtained using 2',3'-cyclic CMP as the substrate. A major protein band of molecular weight approx. 96,000 was found by gel electrophoresis under nonreducing conditions. However, two distinct protein bands of molecular weight 46,000 ± 1400 and 48,000 ± 1400 were observed when the protein sample was reduced with 10 mM-dithiothreitol and subjected to electrophoresis in more restrictive 12-15% polyacrylamide-SDS gels. This molecular weight is lower than that previously reported for the bovine enzyme. Antibodies against the purified human enzyme have been raised in New Zealand white rabbits.     

Amino Acid Sequence Analysis of Reverse Transcriptase Subunits from Avian Myeloblastosis Virus

The NH₂-terminal amino acid sequences of the α and β chains of avian myeloblastosis αβ DNA polymerase were determined by using microsequence analysis in the subnanomole range and were found to be identical up to 17 residues. The common sequence was as follows: Thr-Val-Ala-Leu-His-Leu-Ala-Ile-Pro-Leu-Lys-Trp-Lys-Pro-Asn-His-Thr-. This result provides convincing chemical evidence that the α chain is derived from the NH₂-terminal region of the β chain by proteolytic cleavage, whereas the amino acid composition for these α and β subunits and p32 DNA endonuclease suggests that the latter is derived from the carboxyl-terminal region of the β chain.     

Survival of cohorts in a fluctuating population of the vole Microtus townsendii

Survival patterns of cohorts are described during a population cycle of the vole Microtus townsendii near Vancouver, British Columbia, Canada. A two–year live–trapping study on both enclosed and unfenced populations showed that cohorts during the increase phase of growth lived longest and had the best survival. Smaller voles in the peak density spring cohort had poor survival, but survival increased during the peak density summer. Survival of cohorts in the decline phase breeding season was very poor. The suggestions are made that changes in spacing behaviour may cause changes in cohort survival and that the causes of rapid changes in survival need to be determined.     

Complement-dependent induction of DNA synthesis and proliferation of human diploid fibroblasts

The effects of fresh human serum (FHS) and heat-inactivated human serum (HHS) on the DNA synthesis and proliferation of human diploid fibroblasts were assessed. FHS activated significantly more quiescent fibroblasts to undergo DNA synthesis and proliferation than did HHS. The stimulatory effect occurred consistently over a serum concentration range of 0.1–10%. Using bromodeoxyuridine selective killing techniques, it was shown that this FHS stimulatory effect was on a specific subpopulation of fibroblasts unresponsive to HHS. The involvement of the complement system, and specifically of C1, was shown by the inability of Clq-depleted FHS to support enhanced DNA synthesis whereas Clq-depleted serum reconstituted with purified Clq was effective. Purified Clq did not restore activity when added to heated serum, nor was it mitogenic when tested in basal medium without serum. The addition of purified Clq to fresh serum inhibited the enhancement of DNA synthesis, and at Clq concentrations of 4γ/ml and greater, the fresh serum effects were abrogated. Thus, it appears that binding of the assembled C1 complex to the fibroblast surface was required for FHS-mediated enhancement of fibroblast proliferation, with Clq subcomponent serving as the recognition site. The results from several experiments indicated that antibody was not required for the complement-dependent fibroblast activation. FHS was not cytotoxic, and autologous serum was as effective as allogeneic sera. A 20-fold molar excess of Fab' from pooled human IgG did not alter the FHS effects. FHS from which IgG was more than 99% depleted was still effective. These results suggested an antibody-independent role for complement in the activation of a subpopulation of human diploid fibroblasts.