NADPH-cytochrome P-450 reductase of yeast microsomes.

NADPH-cytochrome c reductase of yeast microsomes was purified to apparent homogeneity by solubilization with sodium cholate, ammonium sulfate fractionation, and chromatography with hydroxylapatite and diethylaminoethyl cellulose. The purified preparation exhibited an apparent molecular weight of 83,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reductase contained one molecule each of flavin-adenine dinucleotide and riboflavin 5′-phosphate, though these were dissociative from the apoenzyme. The purified reductase showed a specific activity of 120 to 140 μmol/min/mg of protein for cytochrome c as the electron acceptor. The reductase could reduce yeast cytochrome P-450, though with a relatively slow rate. The reductase also reacted with rabbit liver cytochrome P-450 and supported the cytochrome P-450-dependent benzphetamine N-demethylation. It can, therefore, be concluded that the NADPH-cytochrome c reductase is assigned for the cytochrome P-450 reductase of yeast. The enzyme could also reduce the detergent-solubilized cytochrome b₅ of yeast. So, this reductase must contribute to the electron transfer from NADPH to cytochrome b₅ that observed in the yeast microsomes.     

Radioimmunoassay for secretin using Nalpha-tyrosylsecretin and [Tyr1]-secretin.

Sensitive radioimmunoassay for secretin was developed by using synthetic preparation of porcine secretin and its related analogs. The secretin-specific antisera with titers ranging 1: 20,000-1 : 150,000 were generated in rabbits against highly purified synthetic secretin. The labeled antigen was prepared by radioiodinating by the chloramine-T method synthetic secretin analog, Nalpha-tyrosylsecretin or [Tyr1]-secretin, both of which were proved to have almost identical immunoreactivities with that of secretin itself. The immunoassay was performed by the double-antibody method using synthetic secretin as standard. The lowest detectable amount of secretin in the present assays was 5-10pg/tube. Human duodenum extract with hot water contained secretin or secretin-like material that shows a parallel displacement curve to the standard in the immunoassay system used. Serum levels of secretin immunoreactivity in man rose up to 250 pg/ml by intraduodenal infusion of HCl and to 800-1,000 pg/ml by i.v. injection of 1 cu/kg of Boots natural secretin.     

The functional site of placental anticoagulant protein: essential histidine residue of placental anticoagulant protein

Placental anticoagulant protein (PAP) rapidly lost its anticoagulant effect due to photooxidation in the presence of methylene blue at pH 7.9 and 8 degrees C. Photooxidized PAP failed to bind the phospholipid vesicle. It seemed unlikely that the protein underwent a change in molecular size during the photooxidation on the basis of its behavior in electrophoresis and gel filtration. Photooxidized PAP had significantly decreased histidine contents, whereas the contents of other amino acids remained essentially unchanged. The peptide, SHLRKV, was included in the functional site of PAP and still showed an anticoagulant activity. On the other hand, the peptide which substituted histidine by alanine, SALRKV, no longer showed the activity. It was shown that the histidine residue is involved in Ca2+ or the phospholipid binding site of the protein.     

A novel cardio-excitatory peptide isolated from the atria of the African giant snail, Achatina fulica

An undecapeptide which potentiates the beat of the ventricle in the African giant snail, Achatina fulica Ferussac, was purified from the atria of the snail. Its primary structure was determined to be H-Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH2. This peptide was found to have excitatory actions not only on the ventricle but also on the penis retractor muscle, the buccal muscle and the identified neurons controlling the buccal muscle movement of Achatina.     

The role of ecdysteroids in the determination of gut-purge timing in the saturniid, Samia cynthia ricini

Under a 12-hr light and 12-hr dark photoperiod, haemolymph ecdysteroid titre of the last(5th)-instar larva of Samia cynthia ricini begins to rise in the early scotophase preceding gut purge, which marks the larval-prepupal transition, to reach a peak titre of 7.6 ng/ml ca. 4.5 hr before gut purge. This profile of ecdysteroid increment is phase-shifted in response to phase shifts of the scotophase immediately before gut purge, in parallel with gut-purge phase shifts, suggesting that ecdysone release is under the control of a circadian clock dictating gut-purge timing. Ecdysone and 20-hydroxyecdysone (10–20 μg), injected no later than 18 hr before the normal gate of gut purge induced well-defined peaks of precocious gut purge ca. 8 hr after injection. Earlier injections caused acceleration of gut purge but the degree of acceleration was unpredictable. These results suggest that the timed surge of ecdysteroids is responsible for the gated occurrence of gut purge and that 18 hr before gut purge larvae acquire the competence to undergo gut purge in a gated fashion provided that they are exposed to a sufficient surge of ecdysteroids.     

Chemotaxonomic significance of ent-kaurene diterpenes in Rabdosia umbrosus varieties

Quantitative identification of six ent-kaurene diterpenes, by reverse phase HPLC, in crude ether extracts of a single leaf of five major Rabdosia umbrosus varieties is described. These diterpenes are significant chemosystematic markers among these plants.     

Effects of sodium chloride,guanidine hydrochloride,and sucrose on the viscoelastic properties of sodium hyaluronate solutions

Effects of sodium chloride (NaCl), guanidine hydrochloride (GuHCl), or sucrose on the viscoelasticity of sodium hyaluronate (NaHA) solutions were studied. NaCl and GuHCl decreased both storage and loss moduli, while sucrose increased both moduli. The critical concentration C* was determined as an inflection point in the plot of zero shear specific viscosity vs concentration for NaHA solutions with and without NaCl, GuHCl, or sucrose. It is suggested that sodium ions or guanidinium ions shield the electrostatic repulsion of NaHA molecules, hence reduce the coil dimension, and C* shifted to higher concentrations. However, sucrose enhances the entanglement coupling between NaHA molecules and retards the disentanglement of molecular chains or promotes to create hydrogen bonds, and then C* for NaHA solutions with sucrose shifts to lower concentrations. This is in agreement with the results of light scattering measurements in the presence of 0.2M NaCl. Both the radius of gyration and hydrodynamic radius of NaHA were reduced in dilute solutions by the addition of sucrose, and added sucrose enhances the interaction between NaHA monomer units. In the case of concentrated NaHA solution, such interactions result to increase the storage and loss moduli because of the enhancement of temporary network formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 23–34, 1999     

A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase.

A soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP). To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA. The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric. The enzyme was used for production of antibodies specific for human TNSALP. Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, upon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyacrylamide gel, indicating that the size difference between the two enzymes is ascribed to N-linked oligosaccharides. The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluorescence studies.     

Involvement of cis-acting repressive element(s) in the 3'-untranslated region of human connective tissue growth factor gene.

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR.     

Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus

To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.