Purification and properties of cytochrome P-450(11beta) from adrenocortical mitochondria.

A cytochrome P-450, which is functional in the steroid methylene 11β-hydroxylation (P-450_11β), has been purified to a protein weight of 85 kg per heme from bovine adrenocortical mitochondria. The purification is accomplished in the presence of deoxycorticosterone as a substrate stabilizer. The procedure involved solubilization of sonicated mitochondrial pellets, ammonium sulfate fractionation, alumina Cγ gel treatment and aniline-substituted Sepharose 4B chromatography.The purified preparation when freed from deoxycorticosterone, has a low spin type absorption spectrum which can rapidly be converted into a typical high spin substrate-bound form by the addition of an 11β-hydroxylatable steroid, either deoxycorticosterone or testosterone. The preparation exhibits high 11β-hydroxylase activity and is free from the cholesterol side-chain cleavage cytochrome P-450 (P-450_scc).The purified P-450_11β, when submitted to SDS-polyacrylamide gel electrophoresis, exhibits a single protein band (molecular weight of 46 kilodaltons) which is clearly distinguished from P-450_scc. As determined by the sedimentation equilibrium method, the molecular weight of the guanidine-treated P-450_11β is estimated to be 43 kilodaltons.     

Chemical synthesis and sequence studies of deoxyribooligonucleotides which constitute the duplex sequence of the lactose operator of Escherichia coli.

We have synthesized the deoxyribooligonucleotide fragments, constituting the sequence of the lac operator of Escherichia coli. Two of these fragments, d(pApApTpTpGpTpTpApT) (nonamer) and d(pApApTpTpGpTpGpApG) (nonamer), corresponding to the 5' termini of lac operator have been synthesized by the phosphodiester method. The remaining four fragments, d(ApCpApApTpT) (hexamer), d(ApTpApApCpApApTpT) (nonamer), d(ApApTpTpGpTpGpApGpCpGpG) (dodecamer), and d(ApApTpTpGpTpTpApTpCpCpGpCpTpC) (pentadecamer), have been synthesized by an improved phosphotriester method. All of the compounds were first characterized by venom and spleen phosphodiesterase digestion to obtain their base composition. The sequence of these oligonucleotides was fully confirmed by the characteristic mobility shifts of their partial venom phosphodiesterase digestion products on two-dimensional homochromatography. A comparative study of the two methods for the synthesis of oligonucleotides has revealed that the phosphotriester method is more convenient than the phosphodiester method because of higher yields and ease of handling large scale preparations.     

Structural studies of lipophorin in insect blood by differential scanning calorimetry and 13C nuclear magnetic relaxation measurements. Location of hydrocarbons

The possible structure of lipophorin in insect blood (hemolymph) was investigated by differential scanning calorimetry (DSC) and 13C nuclear magnetic relaxation studies. The DSC heating curves of intact lipophorins showed endothermic peaks between -3 and 40 degrees C for lipophorins which contain hydrocarbons, whereas no such peaks were observed for lipophorins which do not contain this lipid. Hydrocarbon fractions isolated from the lipophorins showed endothermic peaks similar to those obtained from intact lipophorin in terms of the transition temperatures, the shapes, and the enthalpy changes. 13C spin lattice relaxation times of the (CH2)n resonance of hydrocarbons of intact lipophorin were measured as a function of temperature and revealed that the motions of hydrocarbon chains changed coincidentally with the onset and offset of phase transition. These data suggest the presence of a hydrocarbon-rich region within the lipophorin particles.     

Synthesis and Biological Evaluation of 9-(f-2, c-3-Bishydroxymethyl-r-cyclopropylmethyl)-9H-adenine (A Lower Methylene Homolog of Carbocyclic Oxetanocin) and Related Compounds

Abstract

Adenine (7 and 16), thymine (9a and 18a), and 5-fluorouracil (9b and 18b) involving f-2, c-3-bishydroxymethyl-r-1-cyclopropylmethyl- and t-2 t-3-bishydroxymethyl-r-1-cyclopropylmethyl residues were synthesized, starting from trans-1, 4-dibenzyloxy-2-butene and its cis isomer, respectively. These compounds were evaluated for anti HSV-1 activity.     

Unfolding of CBP21, a lytic polysaccharide monooxygenase,without dissociation of its copper ion cofactor

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu²⁺ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, t_p, for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of t_p in the presence of excess Cu²⁺ was around 75°C, indicating that Cu²⁺ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu²⁺ ⇌ U∙Cu²⁺, where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol⁻¹ in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu²⁺ complex itself.     

Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions

The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me₂SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0–2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO, and that recovered PGCs retained ability to differentiate into functional gametes.     

Microbiological Studies of Coli-aerogenes Bacteria

The presence of glucose-6-phosphate markedly stimulated the anaerobic utilization of glyoxylate by either cell-free extracts or partially purified enzyme preparations of coli-aerogenes bacteria. The enzymic reduction of glyoxylate to glycollate was found to occur in the presence of TPN with the following substrates; glucose-6-phosphate, glucose plus ATP, gluconate plus ATP, glucose-1-phosphate or malate. The data indicated that the reduction of glyoxylate to glycollate was coupled to the oxidation of glucose-6-phosphate via the hexose monophosphate shunt pathway. It was propounded that the operation of the hexose monophosphate oxidative pathway might be controlled by TPN-linked glyoxylic reductase, and the mechanisms of enzymic regulation in microbial respiration were also discussed.     

Production and Isolation of a New Antifungal Antibiotic,Prumycin and Taxonomic Studies of Streptomyces sp., Strain No. F-1028

A newly described species of Streptomyces (named Sm. kagawaensis ATCC No. 21811) isolated from soil was found to produce a new antifungal antibiotic, prumycin, which belongs to amino sugar group. Prumycin was isolated from the fermentation broth by ion-exchange adsorption and gel-filtration methods. This antibiotic inhibited specifically the growth of Sclerotinia sp. and Botrytis sp. on flower pot test with kidney bean leaves and also was effective on the field test with various plants.     

5,7-Dodecadien-1-ol: Candidate for the Sex Pheromone of the Pine Moth

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from Spinacia oleracea. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 86,000 by Ultrogel AcA 34 gel filtration. In the oxidized form, the enzyme had absorption maxima at 278, 388 (Soret band), 573 (α band) and 690 nm, indicating that siroheme is directly involved in the catalysis of nitrite reduction. This absorption spectrum was modified by sulfite, hydroxylamine and cyanide. The enzyme exhibited electron paramagnetic resonance signals with g values of 6.9 and 5.2, which are characteristic of a high spin Fe³⁺ -siroheme in the molecule. These signals disappeared upon the addition of dithionite or nitrite. This isolated enzyme also contained four moles of labile sulfide and 7 g-atoms of iron per 86,000 g of protein.