Exposure of aromatic residues of Streptomyces subtilisin inhibitor. A photo-CIDNP study

Exposure of aromatic residues, Tyr 7, Tyr 75, Tyr 93, His 43, His 106, and Trp 8, was studied by laser-induced photo-CIDNP in the 1H NMR spectrum of Streptomyces subtilisin inhibitor at 360 MHz. Only Tyr 7 and Tyr 75 gave strong CIDNP signals, whereas the rest of the aromatic residues gave no detectable signals in the temperature range 25-55 degrees C. From the temperature dependence data, it is concluded that Tyr 7 is well exposed at all temperatures, whereas the exposure of Tyr 75 increases with temperature, in agreement with the conclusion obtained by other methods. Agreements and discrepancies between the conclusions derived from the CIDNP data and the results so far obtained by other methods are compared for all the aforementioned aromatic residues.     

New synthetic inhibitors of C1r, C1 esterase, thrombin, plasmin, kallikrein and trypsin

p-Guanidinobenzoate derivates were prepared and their inhibitory effects on trypsin, plasmin, pancreatic kallikrein, plasma kallikrein, thrombin, C1r and C1 esterase were examined. Among the various inhibitors tested, 6'-amidino-2-naphthyl-4-guanidinobenzoate dihydrochloride, 4-(beta-amidinoethenyl)phenyl-4-guanidinobenzoate dimethanesulfonate and 4-amidino-2-benzoylphenyl-4-guanidinobenzoate dimethanesulfonate were the most effective inhibitors of trypsin, plasmin, pancreatic kallikrein. plasma kallikrein and thrombin and they strongly inhibited the esterolytic activities of C1r and C1 esterase, and then strongly inhibited complement-mediated hemolysis.     

Effect of a Growth Inhibitor on the Hydroxyproline Level in Cell Wall of Zea Primary Roots

Evidence supporting the view that there is an inverse relationshipbetween the hydroxyproline-protein level in the cell wall andthe ability of a cell to undergo rapid cell elongation was obtained.A growth inhibitor extracted from Zea primary roots acceleratedthe incorporation of radioactivity derived from ¹⁴C-prolineinto the sodium lauryl sulfate (SLS)-insoluble cell wall fraction.However, the inhibitor had no effect on the ratio of hydroxyprolineto proline that was incorporated into the SLS-insoluble fraction. We have discussed what this growth inhibitor may mean in thegeotropic curvature of Zea primary roots. ¹ Present address: Faculty of Education, University of Yamagata,Yamagata 990, Japan (Received May 9, 1981; Accepted August 8, 1981)     

Studies on the interspecies competition between the azuki bean weevil and the southern cowpea weevil IV. Competition between strains

This study analyzes results of interspecies competition between the azuki bean weevil, Callosobruchus chinensis and the southern cowpea weevil, C. maculatus using the four geographical strains of each species.

1. Fifteen combinations of one strain from each species were set up to compete. The strains of C. chinensis, rather than of C. maculatus, characteristically determine the eventual outcomes of competition. Some strains of C. chinensis (jC and kC) invariably won every encounter with C. maculatus. Another strain (nC) always lost its encounters, while one other strain (iC) won some encounters (:cQ and :cQ) and lost others (:tQ and :bQ). For any combination the competition results were deterministic and no reversal in outcome was observed among the replicates.
2. The weakness of strain nC in competition is explained by its characteristics in single species populations, different greatly from other strains of C. chinensis, although it had the similar primary (individual) characteristics as other strains of C. chinensis. Strain iC had the shortest adult longevity in the four strains of C. chinensis although it behaved similar to strain jC at the single species population level. Especially under the food supply schedule of the present experiment the shortness of adult longevity might be the important factor determining the outcome.
3. The relationship of the eight strains based on the individual and single species population characteristics, and that based on the competition results were very similar. Even when the competition results could not be explained by any one or two of these characteristics alone, the outcome would be largely decided by the overall biotic profile made by several characteristics.

     

Hematological and serum biochemical changes with age in term fetuses, offspring and dams in normal Sprague-Dawley rats]

Hematological and serum biochemical values of dams and offspring of Sprague-Dawley rats were measured during late gestation, lactation and postweaning. In dams, slightly low erythrocytic parameters, high platelets and high frequency of neutrophils were seen after parturition although WBC showed no marked changes. On Day 20 of gestation, glucose and triglycerides were extremely high and TP and albumin were low. These changes may be attributed to pregnancy or parturition. In fetuses on Day 20 of gestation and offspring immediately after birth, erythrocytes showed anisocytosis, polychromasia, basophilic stipplings and Howell-Jolly bodies and erythroblasts were found. RBC was low. MCV and MCH were extremely high, compared to adult erythrocyte levels. Hemoglobin and hematocrit slightly decreased before weaning. RBC, hemoglobin and hematocrit increased with age and reached adult levels by Day 56. MCV and MCH values decreased, towards adult levels, until weaning. Platelets rapidly increased and reached adult levels before weaning. WBC increased after birth having higher counts in males than in females on Day 35 and thereafter. Glucose, TP and albumin increased with age and reached adult levels by Day 28. ALP was high in fetuses and changed with age having two peaks similar to those reported in man. Cholesterols gradually increased after birth and had a peak on Day 14. Urea nitrogen, inorganic phosphorus and calcium were slightly high in fetuses and preweaned offspring. Potassium was high in fetuses but no age-related trends were seen in offspring.     

Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action.

In view of the cationic amphipathic structure of tachyplesin I and antiparallel beta-sheet as a general DNA binding motif, DNA binding of the antimicrobial peptide has been examined. Several footprinting-like techniques using DNase I protection, dimethyl sulfate protection, and bleomycin- (BLM-) induced DNA cleavage were applied in this study. Some distinct footprints with DNase I are detected, and also the sequence-specific cleavage mode of the BLM-Fe(II) complex clearly is altered in the presence of tachyplesin I. In addition, methylation of the N-7 residue of guanine situated in the DNA major groove is not entirely inhibited (or activated) by tachyplesin I. The results suggest that tachyplesin I interacts with the minor groove of DNA duplex. Disappearance of the footprints by dithiothreitol-treated tachyplesin I and Ala-tachyplesin strongly suggests a significant contribution of secondary structure containing an antiparallel beta-sheet to the DNA binding of tachyplesin I. This is the first report on DNA interaction with a small peptide which contains a unique antiparallel beta-sheet structure. The mechanism for antimicrobial action of tachyplesin I has also been inferred.     

Maternal Exogastrula-Inducing Peptides (EGIPs) and Their Changes during Development in the Sea Urchin Anthocidaris crassispina

Exogastrula-inducing activity was examined in eggs and embryos of the sea urchin Anthocidaris crassispina at various stages. During fractionation on a column of DEAE-cellulose, the exogastrula-inducing activity was found in the flow-through fraction at all developmental stages. In particular, the activity present in the flow-through fraction of unfertilized eggs represents the presence of maternal exogastrula-inducing peptides (EGIPs). The flow-through fractions from the column of DEAE-cellulose were applied to a column of Sephadex G-100 and the activities in the eluate were assayed. The active low-molecular-weight fraction was obtained in all cases with the exception of pluteus larvae, extracts of which contained another active fraction. Immunoblots of protein samples from eggs and embryos probed with antiserum against EGIP-D indicated that there is a major immunoreactive protein that migrates with an apparent molecular weight of about 6 kDa in all cases with the exception of pluteus larvae, and that there are two major immunoreactive proteins that migrate with apparent molecular weights of 6 kDa and 35 kDa, respectively, in pluteus larvae.     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Kinetics of the hydrolysis of monodispersed dihexanoylphosphatidylcholine catalyzed by bovine pancreatic phospholipase A2: roles of Ca2+ binding and ionizations of amino acid residues in the active site.

Phospholipases A2 (PLA2s) are classified into two groups, I and II, according to differences in the polypeptide-chain length and intramolecular disulfide bondings. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by bovine pancreas PLA2 (Group I) was studied at 25 degrees C and ionic strength 0.2, and the initial velocity data were analyzed by means of the Michaelis-Menten equation. The Michaelis constant, Km, was found to be practically independent of Ca2+ concentration and also of pH value. The latter result indicates no participation of the ionizable groups in the active site in the substrate binding. The pH-dependence curve of the logarithm of the catalytic center activity, kcat, obtained in the presence of a practically saturating amount of Ca2+, showed three transitions ascribable to the participation of three ionizable groups with pK values of 5.00, 8.40, and 9.50. The respective groups were tentatively assigned to the catalytic group His 48, the N-terminal alpha-amino group, and invariant Tyr 52, which is located in close proximity to the imidazole ring of His 48. Deprotonation of His 48 and protonation of Tyr 52 were shown to be essential to the catalysis. The importance of the ionization state of the alpha-amino group was also indicated.(ABSTRACT TRUNCATED AT 250 WORDS)