Purification and properties of human erythrocyte membrane NADH-cytochrome b5 reductase

An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b₅ reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b₅ reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K₃Fe(CN)₆, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b₅ and methemoglobin via cytochrome b₅ when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min^?1 mg^?1 and the turnover number was 40,600 mol ferricyanide reduced min^?1 mol^?1 FAD at 25 °C. The apparent K_m values for NADH and cytochrome b₅ were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b₅ reduced min^?1 mg^?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b₅ reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b₅ reductase.     

Characteristic base sequence patterns of promoter and terminator sites in phi X174 and fd phage DNAs

An overall theory for the evolution of complexity in passerine broadcast song is advanced based on the proposition that new birds should attempt to match the songs of residents. We assume that social systems result from competition between individuals for resources. Individuals which by aggressive interaction have attained some priority of access to a resource (territory, food, or mate) should advertise this. New birds singing in an area will reveal their non-residential status by their song and hence will announce an assymmetry in competition that favours the residents because of their prior knowledge. Thus new birds should learn to match in order to reduce this asymmetry. Counter-selection on residents to reduce the incidence of matching should favour the evolution of song complexity. The degree of complexity should vary directly with the degree of competition. Predictions from these assumptions are advanced.     

Kinetics of the Unrolling of the Second Leaves Detached from Rice Seedlings by Blue Light

Unrolling due to blue light (B) irradiation of the second leavesdetached from 8-day-old rice (Oryza saliva L.) seedlings wassimilar to that reported previously for nondetached leaves.The effect of B was counteracted by irradiation with red light(R). The counteracting effect of R was reversed by subsequentirradiation with far-red light (FR). When the detached leaf was irradiated with B passed througha 1-mm-wide slit 5, 8, 10, 12 or 15 mm from the leaf tip, irradiation10 mm from the leaf tip was the most effective. The effect of a 1 mm-wide-B irradiation 10 mm from the leaftip was counteracted by a 1 mm-wide-R irradiation at the sameposition, but not by irradiations at the other points. The counteractingeffect of R was reversed by a 1 mm-wide-FR irradiation at thesame position. This suggests that the excitation or the reactionof the B photoreceptor(s) is affected directly by the PFR formof phytochrome. The dose-response curve for the unrolling caused by B showeda simple Bunsen-Roscoe relation without two peaks, which differsfrom that for the phototropism in Avena caused by B. (Received August 21, 1980; Accepted December 20, 1980)     

Contribution from the chirality of the growing chain to the enantiomer selectivity in activated-NCA polymerization of α-amino acid N-carboxyanhydride

As a model compound for the growing chain in the activated-NCA type of polymerization of α-amino acid N-carboxyanhydride (NCA), 3-[ω-acetylglycyl-poly(α-amino acid) acyl]-α-amino acid NCA (called the prepolymer) having various degrees of polymerization (DPs) was synthesized by the polymerization of Phe, Val, Glu(OEt), and Asp(OBzl) NCA in the presence of AcGly NCA by the tertiary amine. Activated (S)-Phe, Val, Glu(OEt), and Asp(OBzl) NCA were added to the terminal cyclic group of the corresponding (S)- or (R)- prepolymer, and the enantiomer selectivity in the reaction was investigated. With prepolymers having DPs ranging from 1 to 15, the addition reaction always took place preferentially between species having the same configuration, and the degree of the enantiomer selection increased with increasing DP of the prepolymer. With prepolymers having DP = 1 and 2, we found contributions from the chiral terminal unit and the chiral penultimate unit to the enantiomer selection, respectively. Prepolymer having DP = 5 was shown to take a β-type conformation, which led to higher enantiomer selection; and prepolymers having DP = 10 and 15 were shown to take an α-helix conformation, which led to much higher enantiomer selection than did the β-type conformation. In the present investigation the mechanisms of terminal-unit control, penultimate-unit control and conformational control of the enantiomer selection in the activated-NCA type of polymerization were clearly observed.     

Semisynthesis and properties of some insulin analogs

The trypsin-catalyzed coupling of bovine (Boc)₂-desoctapeptide (B23-B30)-insulin with synthetic octapeptides, H-Gly-X₂-X₃-X₄-Thr-Pro-Lys(Boc)-Thr-OH (X₂ = Phe or Ala, X₃ = Phe or Ala, X₄ = Tyr or Ala), followed by deprotection and purification produced the [Ala^B24, Thr^B30]-, [Ala^B25, Thr^B30]-, and [Ala^B26, Thr^B30]-analogs of bovine insulin in yields of 32, 35, and 32%, respectively. The biological activity of these analogs decreased in the order, normal insulin ([Thr^B30]-bovine insulin) = Ala^B26-insulin > Ala^B25-insulin > Ala^B24-insulin, as assayed for receptor binding and some other biological effects, in contrast with the corresponding Leu-analogs of human insulin, in which the activity decreased in the order, normal insulin > Leu^B24-insulin > Leu^B25-insulin. The affinity to insulin antibodies greatly diminished in both Ala^B24-insulin and Leu^B24-insulin but not in the B25-substituted analogs. The CD spectra of the Leu- and the Ala-analogs were compared with those of normal insulins to show that no apparent correlation seems to exist between the decrease in biological activity and the conformational changes observed in solution. The effects of organic solvents on the peptide-bond equilibrium and on the stability of trypsin are also discussed.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Aplysia punctata added to list of laboratory-cultured Aplysia

The marine opisthobranch molluscAplysia punctata was cultured at the Laboratoire de Biologie Marine in Concarneau, France.A. punctata veligers settled and underwent metamorphosis on the algaLomentaria articulata, but not onUlva spp., Palmaria marina, Laminaria spp. andFucus spp.Research supported by grants from The Arts Foundation and the Lerner Fund for Marine Research of the American Museum of Natural History. We wish to thank Director Yves Legal, College de France for his support and cooperation.     

Fibrin membrane endowed with biological function VI. Immobilization of multienzymes of urea cycle

Four enzymes in urea cycle and inorganic pyrophosphatase were immobilized simultaneously into a matrix of fibrin fiber formed from fibrinogen by the concerted action of thrombin and blood coagulation Factor XIII. The immobilized multienzyme system not only had an ability to carry out urea cycle continuously at least over several hours, but also had a greatly improved efficiency over the corresponding soluble system.     

Comparison of 4 serological tests--complement fixation, neutralization, fluorescent antibody to membrane antigen and immune adherence hemagglutination--for assay of antibody to varicella-zoster (V-Z) virus.

Four tests for antibody to varicella-zoster (V-Z) virus were compared; these were tests of complement fixation (CF), neutralization (NT), fluorescent antibody to membrane antigen (FAMA) and immune adherence hemagglutination (IAHA). Fifty-two sera from patients with varicella and zoster and from recipients of live varicella vaccine were examined by the 4 tests. The CF test was least sensitive, but the antibody titers by the NT, FAMA and IAHA tests were roughly comparable. The IAHA test was the simplest and fastest to perform, and appeared suitable for routine serological assay to V-Z virus. The correlation between the IAHA antibody titer and susceptibility of individuals to clinical varicella was investigated retrospectively using sera obtained during 2 outbreaks of varicella in an institution for children, where all the unvaccinated children had developed varicella symptoms. Most of the 25 pre-exposure sera from unvaccinated children examined by the IAHA test had tiers of less than 1:2. In contrast, all the 23 sera from vaccinated children who did not develop varicella had detectable antibody titers of 1:2 to 1:64. These results indicate that the IAHA titer reflects the susceptibility or resistance of individuals to clinical varicella.