Effect of calmodulin on aldosterone synthesis by a cytochrome P-45011 beta-reconstituted system from bovine adrenocortical mitochondria

Bovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-450(11)beta-reconstituted system in the presence of dilauroylphosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1 nmol/(min X nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6 nmol/(min X nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1 microM, when 1 microM cytochrome P-450(11)beta was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1 microM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-450(11)beta in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20 microM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-450(11)beta. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-450(11)beta in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-450(11) beta.     

High temperature induction of a stringent response in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli

The cellular concentrations of ppGpp in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli were examined, since the thermosensitive RNA synthesis of these mutants is relaxed by an additional mutation in the relA gene. The results showed that ppGpp accumulated extensively in the dnaK(Ts) and dnaJ(Ts) mutants after a temperature shift up, reaching levels of 5 mM and 0.5 mM, respectively. This unusual accumulation of ppGpp was suppressed by the relA1 mutation, implying that it results from induction of a stringent response in these mutants at a nonpermissive temperature.     

Flowering Response in Relation to C and N Contents of Pharbitis nil Plants Cultured in Nitrogen-Poor Media

Flowering of seedlings of Pharbitis nil, strains Violet andTendan, cultured in modified White's medium, was promoted bymedium dilution, the critical dark period being shortened byabout 15 min. Dilution of the N source alone was enough to causethe medium-dilution effect. Dilution of the culture medium duringthe day before and on the day of exposure to the dark-period(a total of two days) caused the maximum dilution effect. TheC and N contents of the cotyledons and of the shoot apices changedrapidly in response to medium dilution. In 1/2-strength White'smedium with 1/1,000 strength NO₃^– which was most effectivefor flower promotion, the C-N ratio was highest. In 1/2-strengthmodified White's medium, in which flowering was lowest withthe longest critical dark period, the C-N ratio was lowest.Thus, there is a close relation between flowering response andthe C-N ratio in cotyledons or shoot apices of Pharbitis nil. (Received September 14, 1984; Accepted January 26, 1985)     

Proteolysis of liver acetyl coenzyme A carboxylase by cathepsin B

Proteolysis of acetyl-CoA carboxylase was examined with cathepsin B. When chicken liver acetyl-CoA carboxylase was incubated with cathepsin B at pH 6.3, the native 220-kDa polypeptide was primarily cleaved into two polypeptides of 125 and 115 kDa, and further degraded to polypeptides of 100-50 kDa.     

Transient glucose intolerance during attacks of thyrotoxic periodic paralysis

In a 19-year-old Japanese male (case 1) with thyrotoxic periodic paralysis (TPP), an increase of plasma glucose concentration together with abnormally high levels of serum immunoreactive insulin (IRI) was observed preceding a spontaneous attack of paralysis. Therefore, the plasma glucose, glucagon, epinephrine, norepinephrine, serum IRI, growth hormone and cortisol levels, and the erythrocyte insulin receptors were measured in case 1 and a 40-year-old Japanese male (case 2) with TPP during attacks of paralysis induced by prolonged glucose loading. In case 1, the serum IRI concentration was elevated to the extraordinarily high level of 655.0 microU/ml at the beginning of paralysis, and at that time, the plasma glucose concentration was 147 mg/dl. However, when paralysis was not induced by a similar glucose loading during methimazole treatment, the serum IRI and plasma glucose levels at the corresponding time after glucose loading were 20.9 microU/ml and 87 mg/dl, respectively. Furthermore, the affinity of the erythrocyte insulin receptors was decreased during the attack. In case 2, plasma glucose and serum IRI concentrations were increased in accordance with the initiation of paralysis although the blood levels of hormones counteracting insulin were not significantly changed. These findings suggest that there is something interacting with the normal action of the insulin in the early phase of paralysis.     

Probing stability and dynamics of proteins by protease digestion. I: Comparison of protease susceptibility and thermal stability of cytochromes c

Protease susceptibility of homologous proteins in their native conformations was studied. This work aims to establish a broad and quantitative basis for the utilization of protease digestion to analyze the local stability of native proteins. Using high-performance liquid chromatography (HPLC) the time course of the proteolytic degradation of intact proteins was quantitatively traced. Rapid separation of peptide fragments with HPLC made possible the elucidation of sequential digestion originating from the cleavage at a very few sites which are locally unstable in the protein structure. Using four serine proteases, chymotrypsin, trypsin, elastase and subtilisin BPN', we found some common trends in proteolysis for a group of proteins of the cytochrome c family. By comparing of the proteolysis and thermal denaturation with ten homologous cytochromes c extracted from horse, beef, Candida krusei, Saccharomyces cerevisiae, chicken, tuna, pigeon, rabbit, dog and rat, protease susceptibility was related to locally unfolding states intrinsic to the native conformation.     

Fetal hemoglobin variants in 80,000 Japanese neonates: high prevalence of Hb F Yamaguchi (AγT 80 Asp→Asn)

Summary A population-based screening of newborns for the structural variants of fetal hemoglobin was carried out in Osaka Prefecture, Japan, by isoelectric focusing of globin chains using dried blood on filter paper. Of 80,000 newborns, 18 had globin variants and 55 had globin variants. The incidence of globin variants (1/1,455) was much higher than that of globin variants (1/4,444). Structural studies were then carried out on the abnormal globins in 36 samples, and revealed that 25 of them were Hb F Yamaguchi (^AT 80 AspAsn). The prevalence of this variant in Japanese was estimated to be as high as one per 2,100.     

Circadian rhythms of testosterone-dependent behaviors,crowing and locomotor activity,in male Japanese quail

Summary An apparatus was devised to record crowing (mate calling by males) together with locomotor activity and recorded data was analyzed by several methods for rhythm analysis. Crowing and locomotor activity of Japanese quail held on long days were recorded during sexual development as estimated from circulating gonadotropins and testosterone. Both behaviors were testosterone-dependent but commencement of crowing preceded the increase in locomotor activity. When the two behaviors attained their maximum levels, crowing showed consistent daily rhythms in which two peaks were apparent, a major one at the onset of light and a broader one 8 hours later. Locomotor activity also showed a clear daily rhythm with a peak between the two peaks of crowing rhythm suggesting a fixed phase relationship between the two rhythms.Both rhythms free-ran under constant dim light with periods shorter than 24 h. They persisted in birds which had been castrated and then supplied with exogenous testosterone via implanted Silastic capsules. The durations of both rhythms were quite comparable to each other and they maintained a fixed phase relationship similar to that found under LD cycles.The results indicate that testosterone is essential for the induction of crowing and for the enhancement of locomotor activity but the formation of the rhythms in behavior was strictly dependent on a circadian oscillatory mechanism.Abbreviations LH luteinizing hormone - FHS follicle-stimulating hormone - LD light-dark - LDim light-dim light     

Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits

E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding. The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents. The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.     

Spirulina ferredoxin-NADP+ reductase. Further characterization with an improved preparation

The preparation procedure for Spirulina ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1.18.1.2, FNR) was improved by adding protease inhibitors, phenylmethylsulfonylfluoride (PMSF) and EDTA, through the whole process of preparation and by introducing an affinity chromatography step on Blue Sepharose CL-6B. The addition of the inhibitors largely prevented the formation of the minor component (FNR I), and the affinity gel chromatography simplified the preparation process, shortening the exposure period of FNR to proteolysis. However, complete removal of the heterogeneity of FNR found at the amino (N)-terminal region was not achieved even by applying the new method. The affinity chromatography on the Blue Sepharose gel was also effective in purifying spinach FNR. The affinity of this gel for Spirulina FNR was compared with that for the enzyme derived from spinach leaves. The spinach enzyme had a higher affinity than the Spirulina one. Both enzymes showed the highest affinities to Blue Sepharose at 20--30 mM NaCl concentration. The N-terminal sequence analysis revealed that there was 4 forms, which were probably modifications produced by exopeptidase action during the preparation, or even in the living cells. The longest component gave the N-terminal sequence Ala-Lys-Thr-Asp-Ile-Pro-Val-Asn-Ile-Tyr-. The others lacked amino acids successively one by one from the N-terminus. In contrast, the carboxyl(C)-terminal residues of all 4 FNR forms were tyrosine. The probable C-terminal sequence was predicted to be -Trp-His-Val-Gln-Thr-Tyr based on a study of a cyanogen bromide peptide.