Purification and Properties of Prostaglandin H-E Isomerase from the Cytosol of Human Brain: Identification as Anionic Forms of Glutathione S-Transferase

Abstract: Prostaglandin H-E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl-Sephar-ose chromatography, gel filtration on a BioGel P-100 column, GSH-agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were ∼5.4. The enzymes catalyzed selective conversion of prostaglandin H₂ to prostaglandin E₂. The K _m values for prostaglandin H₂ of peaks 1 and 2 were 147 and 308 μ M , respectively, and the V _max values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S -transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H-E isomerase. Prostaglandin H-E isomerase activity of peak 2 enzyme was competitively inhibited by 1-chloro-2,4-dinitrobenzene, a substrate of glutathione S -transferase. These results suggested that prostaglandin H-E isomerases in human brain cytosol were identical with anionic forms of glutathione S -transferase.     

Postglacial environmental change of the Pacific Ocean off the coasts of central Japan

Downcore changes in microfossil assemblages and oxygen isotope ratios in three piston cores recovered from the Northwestern Pacific, off central Japan, show that the subtropical Kuroshio front was located to the south of C-4 core site (Lat. 33° N) during the last glacial. The front then advanced northward, passing over the C-4 site and the C-6 site (34.6° N) at about 13 ka and 10 ka, respectively, and reached the C-1 core site (36° N) at about 7 ka. After 5.5 ka it retreated to the area between the C-1 and C-6 sites. A brief but significant cold event, the readvance of the cold Oyashio Current, is recognized between 11 and 10 ka in the two northern cores, but the current did not reach the southern C-4 site. A contemporaneous cold event is known in the North Atlantic, and the cooling was probably a global phenomenon likely to be associated with lowering of sea level. Contamination of isotopically light water is apparent between 14 and 11 ka in the marked change in isotopic composition of benthic foraminifers. Oxygen isotope ratios of planktonic foraminifers show that prior to the advance of the Kuroshio front, the surface water at these core sites was isotopically lighter than the Kuroshio water at that time.     

Hg2+-induced contracture in mechanically skinned fibers of frog skeletal muscle

In mechanically skinned fibers of the semitendinosus muscle of bullfrogs, we examined the role of membrane sulfhydryl groups on Ca2+ release from the sarcoplasmic reticulum (SR). Hg2+, a sulfhydryl reagent (20-100 microM), induced a repetitive contracture of skinned fibers, and this contracture did not occur in skinned fibers in which the SR had been disrupted by treatment with a detergent (Brij 58). Procaine (10 mM), Mg2+ (5 mM), or dithiothreitol (1 mM) blocked the Hg2+-induced contracture. Ag+ or p-chloromercuribenzenesulfonic acid produced similar contractures to that induced by Hg2+. We conclude that Hg2+ releases Ca2+ from SR of a skinned fiber by modifying sulfhydryl groups on the SR membrane, and suggest that the Ca2+ released by Hg2+ may trigger a greater release of Ca2+ from SR to develop tension.     

Callus formation from protoplasts of culturedSpinacia oleracea cells

Cell suspensions were initiated from plumule derived calli ofSpinacia oleracea. Some of these cell lines could be maintained in culture for at least three years without a reduced growth rate. A high yield of protoplasts was obtained from the cell suspensions. When protoplasts were cultured in Murashige and Skoog medium with naphthaleneacetic acid and 6-benzyladenine, cell wall formation was observed after three days. The cultured protoplasts produced numerous cell-clusters within two weeks. However only protoplasts isolated from suspensions which were in a rapidly dividing phase were able to divide with a high frequency and give rise to callus colonies.     

Effect of temperature and Zn2+ on isometric contractile properties and electrical phenomena of frog (Rana) and Xenopus skeletal muscle fibers

Effects of temperature and Zn2+ on the isometric contractile properties of toe muscle fibers of Rana catesbeiana and Xenopus laevis were studied. The maximum twitch tension almost doubled when the temperature was lowered from 20 to 4 degrees C in Rana muscles but not in Xenopus muscles, although the duration of action potential in Xenopus muscle was increased slightly more than that seen in the Rana species. The maximum rate of rise of tension was greater in Xenopus muscle than in the Rana muscle, at 20 degrees C. The prolongation of the time-to-peak tension following exposure to low temperature (4 degrees C) was more pronounced in Rana than in Xenopus muscles. These results suggest that the speed of release and reuptake of Ca2+ by the sarcoplasmic reticulum (SR) differs in Rana and Xenopus muscles and that these factors may be related to differences in the SR and the T-tubular morphology. In Rana muscles, Zn2+ prolonged the falling phase of the action potential and potentiated the twitch tension. In Xenopus muscles, Zn2+ marginally prolonged the duration of action potential and the twitch tension was not markedly potentiated. These results indicate that Zn2+ potentiates the twitch by prolonging the action potential and that Rana muscles are more sensitive to the effects of Zn2+.     

Structural changes of alpha2- and ovomacroglobulins

The plasma α₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α₂-macroglobulin.     

Studies on the biological activity of an insulin-stimulating peptide from a tryptic digest of bovine serum albumin

Summary A two-chain polypeptide, which corresponds to amino acid residues 115–143 and 144–184(185) of bovine serum albumin, connected to each other by a disulfide bridge, potentiated the effects of insulin on glucose transport and glucose metabolism in isolated rat adipocytes. Although the peptide alone had little activity, it shifted the concentration-response curves of insulin-stimulated D-[I-¹⁴C]glucose oxidation, 2-deoxyglucose transport, and lipid synthesis from D-[U-¹⁴C]glucose to lower insulin concentrations. It also increased the maximal responses of these parameters to insulin. However, it did not affect insulin binding to adipocytes. The peptide protected insulin considerably from degradation, but this effect alone cannot account for its effect in increasing the maximal responses to the hormone, and even when degradation of a submaximal concentration of insulin was suppressed by bacitracin, the peptide still had an enhancing effect. These results suggest not only that the peptide influences a step distal to receptor-mediated insulin binding but also that inhibition of insulin degradation alone cannot explain its total effect.The peptide lost its insulin-stimulating activity completely when it was further digested with V8 or lysinespecific endopeptidase, or when it was reduced and then carboxamidomethylated or oxidized with performic acid. Similar active tryptic fragments were obtained from human and rat albumins.Insulin-stimulating peptides should be useful in studies on the mechanisms of insulin action including both the sensitivities and responsiveness of target cells to the hormone.Abbreviations ISP insulin-stimulating peptide - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - HPLC high-performance liquid chromatography - SDS sodium dodecyl sulfate     

Step-wise thermal denaturation of cobrotoxin,a snake venom neurotoxin fromNaja naja atra: A proton nuclear magnetic resonance study

Temperature dependence of proton nuclear magnetic resonance spectra has been followed for cobrotoxin, a postsynaptic neurotoxin fromNaja naja atra venom. Several aromatic amino-acid residues, including the functionally essential Trp-29 located at the tip of the central loop of the molecule, have been found to undergo a thermal structural transition above the global thermal denaturation temperature. It is suggested that a local structure around these residues behaves somehow independently of the rest of the molecule, and that such structural organization may be favorable for a conformational change of a neurotoxin molecule on binding to acetylcholine receptor.     

Male emergence timing and mating success in the funnel-web spider,Agelena limbata (Araneae: Agelenidae)

Field observations on the relationship between male mating success and emergence timing in the funnel-web spider,Agelena limbata, were conducted.Agelena limbata is an annual species and adult males appear slightly earlier than adult females in July. As males deposit a copulatory plug at the female epigynum after copulation, copulation with virgin females is important to males. The number of copulations in males with virgin females, which strongly correlates with the longevity of males and the number of females that males courted, did not correlate with the emergence timing of males. Early emerged males and females were significantly larger in size than later ones, but the correlation coefficient between the emerged date and the cephalothorax width was not strong. Males that emerged earlier did not have any advantage in copulating with larger and more fecund females. Furthermore, virgin females first copulated on average 7.9 days after their final molt and the mortality rate of adult males increased after the final molt. These factors may favor the smaller degree of protandry in male emergence timing inA. limbata.