Comparative study of two arginine ester hydrolases, E-I and E-II from the venom of Crotalus ruber ruber (red rattlesnake)

1. Two arginine ester hydrolases, E-I and E-II from the venom of Crotalus ruber ruber were isolated and characterized. 2. E-I and E-II have molecular weights of 32,000 and 33,000, and isoelectric points of 5.2 and 4.6, respectively. 3. E-I and E-II are active upon the glandular kallikrein substrate, but neither enzyme was shown to have plasma kallikrein substrate hydrolytic activity. 4. E-I has minimal fibrinogen-clotting activity. It was found to induce clotting by catalyzing the hydrolysis of only the A fibrinopeptide from the A alpha-chain of fibrinogen.     

Purification and biochemical characterization of two soluble alpha-mannosidases from Candida albicans.

Two soluble alpha-mannosidases, E-I and E-II, were purified from C. albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42 degrees C. Apparent Kmvalues for hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside by E-I were 0.83 microM and 2. 4 mM, respectively. Corresponding values for E-II were 0.25 microM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-alpha-D-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+and Ca2+in the range of 0.5-2 mM. At the same concentrations, Mg2+was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved alpha-1,6 and alpha-1,3 linkages, respectively.     

Two active forms of valyl-tRNA synthetase from Bacillus subtilis: alteration related to the early stages of sporulation

Two enzymatically active forms, E-I and E-II, of valyl-tRNA synthetase [EC 6.1.1.9] from cells at various stages in the life cycle of Bacillus subtilis 168 LTT, germinated cells, vegetative cells (t-0.5), sporulating cells (t0, t1, t3, and t4), forespores and mature spores, were analyzed by hydroxyapatite column chromatography. The E-II activity was detected in the main fraction of valyl-tRNA synthetase during the life cycle of B. subtilis 168 LTT. The high activity of E-II at t0 decreased rapidly in the stationary and sporulating phases. On the other hand, the E-I activity increased in the early sporulating stage and was about twofold higher at t3 than at t0. After t3, this activity also decreased rapidly and was not detected in forespores and mature spores. The relative amount of E-I at t0 was 3.4% of the total valyl-tRNA synthetase activity eluted from the hydroxyapatite column, 12.9% at t1 and 29.2% at t3, but it was less than 10% at t4 and in germinated cells. The alteration in E-I and E-II activities was also observed in cells of B. subtilis NIG 1121 (spo+), W23 and 168W, but not in any asporogenous mutant strain studied. These results show that the alteration in the valyl-tRNA synthetase activity appears only during the early stages of sporulation and is closely related to the sporulation of B. subtilis.     

Conversion of α1,2-mannosidase E-I from Candida albicans to α1,2-mannosidase E-II by limited proteolysis

Previous studies demonstrated the presence in Candida albicans ATCC 26555 of two soluble α1,2-mannosidases: E-I and E-II. In contrast, in the C. albicans CAI-4 mutant only E-I was detected and it could be processed by a membrane-bound proteolytic activity from the ATCC 26555 strain, generating an active 43 kDa polypeptide. Here, α1,2-mannosidase E-I from strain ATCC 26555 was purified by conventional methods of protein isolation and affinity chromatography in Concanavalin A-Sepharose 4B. Analytical electrophoresis of the purified enzyme revealed two polypeptides of 52 and 23 kDa, the former being responsible for enzyme activity as revealed by zymogram analysis. Time course proteolysis with an aspartyl protease from Aspergillus saitoi, converted α1,2-mannosidase E-I into an active polypeptide of 43 kDa which trimmed Man₉GlcNAc₂, generating Man₈GlcNAc₂ isomer B and mannose. Trimming was inhibited preferentially by 1-deoxymannojirimycin. Both, the molecular mass and the enzyme properties of the proteolytic product were identical to those described for α1,2-mannosidase E-II therefore supporting the notion that E-I is the precursor of E-II.     

Processing of the Man(10)GlcNAc (M(10)) oligosaccharide by alpha-mannosidases from Candida albicans

The hydrolysis of Man(10)GlcNAc (M(10)) by purified alpha-mannosidases and its further processing by a mixed membrane preparation from Candida albicans were studied. Incubation of the oligosaccharide with purified alpha-mannosidases I (E-I) or II (E-II) from C. albicans released 1 and 2 mol of mannose per mol of M(10), respectively. This treatment converted M(10) into an acceptor substrate of further mannose residues from GDP-Man as catalyzed by membrane-bound mannosyltransferases. Elongation of E-I- or E-II-trimmed M(10) yielded a low molecular mass product (14-17 mannose residues added), and in the case of E-II, a minor amount of an additional product of a higher molecular mass. Our results indicate that purified alpha-mannosidases participate in N-glycan processing in C. albicans.     

Chimeric analysis of the small GTPase RacE in cytokinesis signaling in Dictyostelium discoideum

RacE is a small GTPase required for cytokinesis in Dictyostelium discoideum. To investigate RacE's potential binding and signaling interfaces that allow its function in cytokinesis, 10 different chimeras were created between RacE and the closely related small GTPase, RacC. RacE/RacC chimeras, containing various combinations of four RacE regions, E I-IV: E-I (aa 1-67), E-II (aa 68-124), E-III (aa 125-184), and E-IV (aa 185-223), were tested for their ability to rescue the multinucleated, cytokinesis-defective phenotype of RacE null cells grown in suspension. Regions E-II and E-IV were essential but not sufficient for the rescue of RacE null cells. These two regions, in combination with either region E-1 or E-III, resulted in rescue. Results presented here suggest that region E-II contains a crucial, yet incomplete, binding site. Regions E-I or E-III separately provide additional, necessary elements for RacE's function. The extended E tail of RacE (E-IV) may act as a 'sensor' of the bound nucleotide state of RacE and facilitate GDP to GTP exchange (possibly through interactions with a GEF molecule), thereby resulting in activation of RacE. This study provides new evidence for small GTPases engaging several distinct protein interfaces to mediate signaling in various cellular processes.     

Partial purification and property of pyridoxine (pyridoxamine)-5′-phosphate oxidase isozymes from wheat seedlings

Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes.     

Spontaneous development of anti-hepatitis B virus envelope (anti-idiotypic) antibodies in animals immunized with human liver endonexin II or with the F(ab')2 fragment of anti-human liver endonexin II immunoglobulin G: evidence for a receptor-ligand-like relationship between small hepatitis B surface antigen and endonexin II.

In a previous study, we have identified endonexin II (E-II) on human liver plasma membranes as a specific, Ca(2+)-dependent, small hepatitis B surface antigen (HBsAg)-binding protein. In this article, we describe the spontaneous development of anti-HBs antibodies in rabbits immunized with native or recombinant human liver E-II and in chickens immunized with the F(ab')2 fragment of rabbit anti-human liver E-II immunoglobulin G. Anti-HBs activity was not observed in rabbits immunized with rat liver E-II. Cross-reactivity of anti-E-II antibodies to HBsAg epitopes was excluded, since anti-HBs and anti-E-II activities can be separated by E-II affinity chromatography. The existence of an anti-idiotypic antibody is further demonstrated by competitive binding of human liver E-II and this antibody (Ab2) to small HBsAg, suggesting that Ab2 mimics a specific E-II epitope that interacts with small HBsAg. In addition, it was demonstrated that anti-HBs antibodies developed in rabbits after immunization with intact human liver E-II or in chickens after immunization with F(ab')2 fragments of rabbit anti-human liver E-II immunoglobulin G recognize the same epitopes on small HBsAg. These findings strongly indicate that human liver E-II is a very specific small HBsAg-binding protein and support the assumption that human liver E-II is the hepatitis B virus receptor protein.     

Effects of three strains of intestinal autochthonous bacteria and their extracellular products on the immune response and disease resistance of common carp,Cyprinus carpio

The study isolated three strains of intestinal autochthonous bacteria Aeromonas veronii BA-1, Vibrio lentus BA-2, and Flavobacterium sasangense BA-3 from the intestinal tract of the common carp (Cyprinus carpio). To reveal the effects of these three strains of bacteria on the innate immunity of carp, the lysozyme, complement C3, total serum protein, albumin and globulin levels, respiratory burst activity, phagocytic activity by blood leucocytes and the expression of IL-1b, lysozyme-C, and TNF-α were examined after feeding with seven different diets for up to 28 days. Also the survival of carp against Aeromonas hydrophila was challenged for 14 days. The carp were fed seven different diets: one control, three diets supplemented with 1 × 10⁸ cell g⁻¹ of carp intestinal bacteria BA-1 (Group D-I), BA-2 (Group D-II) and BA-3 (Group D-III), and three diets supplemented with extracellular products FA-1 (Group E-I), FA-2 (Group E-II) and FA-3 (Group E-III) which were corresponding to the strains BA-1, BA-2, and BA-3, respectively, up to 28 days. For groups D-I, D-III, E-I and E-III, the innate immune parameters of carp were significantly increased, the expression of three immune-related genes in blood was significantly up-regulated examined during 7, 14, and 21 days of feeding, and the survival rate was improved. The study indicates that the two isolated intestinal autochthonous bacteria A. veronii BA-1 and F. sasangense BA-3 could positively influence immune response and enhance disease resistance of carp against A. hydrophila infection.     

Synthesis of delta(E)Phe-containing tripeptide via photoisomerization and its conformation in solution

A new synthetic route to (E)-beta-phenyl-alpha,beta-dehydroalanine (delta(E)Phe)-containing peptide was presented via photochemical isomerization of the corresponding (Z)-beta-phenyl-alpha,beta-dehydroalanine (delta(Z)Phe)-containing peptide. By applying this method to Boc-Ala-delta(Z)Phe-Val-OMe (Z-I: Boc, t-butoxycarbonyl; OMe, methoxy), Boc-Ala-delta(E)Phe-Val-OMe (E-I) was obtained. The identification of peptide E-I was evidenced by 1H-nmr, 13C-nmr, and uv absorption spectroscopy, elemental analysis, and hydrogenation. The conformation of peptide E-I in CDCl3 was investigated by 1H-nmr spectroscopy (solvent dependence of NH chemical shift and difference nuclear Overhauser effect). Interestingly, peptide E-I differed from peptide Z-I in the hydrogen-bonding mode. Namely, for peptide Z-I, only Val NH participates in intramolecular hydrogen bonding, which leads to a type II beta-turn conformation supported by hydrogen bonding between CO(Boc) and NH(Val). On the other hand, for peptide E-I, two NHs, delta(E)Phe NH and Val NH, participate in intramolecular hydrogen bonding. In both peptides, a remarkable NOE (approximately 11-13%) was observed for Ala C(alpha) H-deltaPhe NH pair. Based on the nmr data and conformational energy calculation, it should be concluded that peptide E-I takes two consecutive gamma-turn conformations supported by hydrogen bonding between CO(Boc) and NH(delta(E)Phe), and between CO(Ala) and NH(Val) as its plausible conformation.     

Receptor site analysis using neurosensory responses of the boll weevil to analogs of the cyclohexylideneethanol of its aggregation pheromone

The active sites of II neurons (Dickens, 1990c) in both sexesof the boll weevil, Anthonomus grandis Boh., were investigatedusing structure—activity relationship studies. Hectrophysiologicalresponses of antennal olfactory receptors in male and femaleboll weevils were recorded in response to components II andIII of its aggregation pheromone and various functional, geometric,and 2-fluoroalkenyl analogs. Dose—response curves forelectroantennogram responses of both sexes to both the 2-fluoroanalog of II (E-II-F) and II (Z-II) were essentially identical.Correspondingly, dose—response curves for the geometricisomer of II (E-II), and its 2-fluoro analog (Z-II-F), had similarshapes but responses to E-II were significantly greater at severaldoses. Responses of single neurons associated with type I sensillato 1 µg stimulus loads of various analogs of II showedsignificant differences in the numbers of impulses elicitedby each during a 500 ms stimulation period: Z-II > E-II-F> III--F, III > Z-II-F, E-II, IV. These results demonstratedthe importance of the cis geometry between the dimethyl groupat the 3-position on the six-carbon ring, and the functionalmoiety at the 1-position. The limited activity of E-II and Z-II-Fon II neurons indicates these compounds may be stimulating otherreceptor types, since EAGs to these compounds were large. Dose—responsecurves for an II neuron were similar for Z-D and E-II-F, aswere curves for III and III--F, indicating that the fluorineatom at the 2-position had little effect on receptor binding.Our data support a previous hypothesis that II neurons haveseparate receptors sites for pheromone components II and III.     

Aerobically generated CO(2) stored during early exercise.

Previous studies have shown that a metabolic alkalosis develops in the muscle during early exercise. This has been linked to phosphocreatine hydrolysis. Over a similar time frame, the femoral vein blood pH and plasma K(+) and HCO(-)(3) concentrations increase without an increase in PCO(2). Thus CO(2) from aerobic metabolism is converted to HCO(-)(3) rather than being eliminated by the lungs. The purpose of this study was to quantify the increase in early CO(2) stores and the component due to the exercise-induced metabolic alkalosis (E-I Alk). To avoid masking the increase in CO(2) stores by CO(2) released as HCO(-)(3) buffers lactic acid, the transient increase in CO(2) stores was measured only for work rates (WRs) below the lactic acidosis threshold (LAT). The increase in CO(2) stores was evident at the airway starting at approximately 15 s; the increase reached a peak at approximately 60 s and was complete by approximately 3 min of exercise. The increase in CO(2) stores was greater, but the kinetics were unaffected at the higher WR. Three components of the change in aerobically generated CO(2) stores were considered relevant: the carbamate component of the Haldane effect, the increase in CO(2) stores due to increase in tissue PCO(2), and the E-I Alk. The Haldane effect was calculated to be approximately 5%. Physically dissolved CO(2) in the tissues was approximately 30% of the store increase. The remaining E-I Alk CO(2) stores averaged 61 and 68% for 60 and 80% LAT WRs, respectively. The kinetics of O(2) uptake correlated with the time course of the increase in CO(2) stores; the size of the O(2) deficit correlated with the size of the E-I Alk component of the CO(2) stores. We conclude that a major component of the aerobically generated increase in CO(2) stores is the new HCO(-)(3) generated as phosphocreatine is converted to creatine.     

Aminooxyphosphonates as Slow Binding Inhibitors of Aspartate and Alanine Aminotransferases from Porcine Heart

Abstract

Aminooxymethylphosphonic (AOMP), 1–aminooxyethylphosphonic (1-AOEP) and 2-aminooxyethyl-phosphonic (2-AOEP) acids have been synthesised and were found to be potent slow binding inhibitors of aspartate- and alanine-aminotransferases with K_i ranging from nanomolar to micromolar values. The half-life of the inhibited complexes varied from 8 min (AspAT-2-AOEP) to 11 h (AspAT-AOMP). Kinetic analysis of the interaction of both enzymes with AOMP suggested the formation of an E-I complex in a single slow binding process. In the case of other compounds, attempt to discriminate between a single- or a double-step mechanism, consistent with an E-I intermediate followed by a slow E-I to E-I* isomerisation process could not be clearly resolved. Spectral studies of the complex formed between PLP-bound enzyme and the aminooxy compound resulted in a shift from 362 nm, the absorption maximum of the native enzyme, to 380 nm, characteristic of the oxime produced. The kinetic parameters for aminooxyphosphonates were compared to those for their carboxylic and aminophosphonic analogues.     

Diet-induced hyperlipidemia in cattle modifies the intrafollicular cholesterol environment, modulates ovarian follicular dynamics, and hastens the onset of postpartum luteal activity.

Three experiments were conducted with female cattle during the postpartum period and during the estrous cycle to examine the effects of diet-induced hyperlipidemia on lipoprotein-cholesterol and triglyceride (TG) metabolism, ovarian follicular dynamics, and incidence of postpartum luteal activity. Dietary-lipid effects were examined independently of metabolizable energy intake. Feeding a high-lipid (HL) diet (8% total lipid) for the first 3 wk of the puerperium (Experiment 1) increased (p less than 0.0001) the concentration of total cholesterol and high-density lipoprotein-cholesterol (HDL-CH), but not TG, within follicular fluid (FF) of estrogen-active (E-A) and inactive (E-I) follicles. Increases (1.4- to 1.7-fold) were similar in proportion to those observed in peripheral blood serum, but absolute concentrations were about 45% of that in serum. Greater than 95% of the cholesterol in FF was HDL-CH, with a greater (p less than 0.06) proportion of HDL-CH sequestered by E-A compared to E-I follicles. The HL diet increased (p less than 0.06) the number of medium-sized (3.1-9.9 mm) follicles present at ovariectomy 19-21 days postcalving and increased (p less than 0.03) concentrations of FF androstenedione in E-I follicles 5-fold. Granulosa cells from preovulatory follicles (Experiment 2) of heifers fed HL diets secreted 2.1- to 3.5-fold greater (p less than 0.03) quantities of pregnenolone and progesterone in vitro. Finally, feeding HL supplements to postpartum range cattle for 30 days increased (p less than 0.05) the incidence of ovarian luteal activity by 18% (Experiment 3). Shifts in lipid metabolic status modify reproductive potential in cattle, independently of dietary energy intake.     

Structural aspects of the exudate from the fruit of Chorisia speciosa St. Hil

Mature fruit of Chorisia speciosa yield an exudate (E-I) following mechanical injury. The polysaccharide contains rhamnose, arabinose, xylose, mannose, glucose, galactose and glucuronic acid in molar ratios of 20:11:1:3:2:40:23. The main chain of the structure is composed by beta-galactopyranosyl units linked (1 --> 3) and (1 --> 6) as indicated by NMR spectra and methylation data. Arabinosef and rhamnose are terminal residues. In order to compare E-I with the polysaccharides from the fruit mesocarp, the latter was submitted to different extractions. The water fraction contains rhamnose, arabinose, xylose, mannose, glucose, galactose and uronic acid in molar ratios of 18:4:1:2:3:44:28. It was treated with CTAB yielding a precipitate which was decomplexed with NaCl, giving four fractions. The fraction obtained using 0.15 M NaCl had a quantitative composition similar that of E-I.     

Esterase electrophoretic polymorphism of human and animal strains of Clostridium perfringens.

Esterase electrophoretic polymorphism in human and animal strains of Clostridium perfringens was studied by using polyacrylamide-agarose gel electrophoresis. Five types of esterases, designated E-I to E-V and defined by their hydrolytic specificities toward five synthetic substrates, were found in protein extracts of bacteria grown without glucose (glucose-containing media allowed only the expression of esterase E-I). Mobility variants of esterase E-I, which hydrolyzes alpha- and beta-naphthyl acetates and butyrates, were used as a basis for the distribution of strains into 11 zymogroups. When all five types of esterases and their electrophoretic variants were considered, 77 electrophoretic types (ETs) could be described for the 89 strains tested. Animal strains did not constitute a distinctive subpopulation, as revealed by their distribution in the zymogroups and by clustering analysis. Statistical analysis also emphasized the importance of esterase E-IV (which hydrolyzes only naphthyl acetates) and esterase E-V (which hydrolyzes only alpha-naphthyl acetate) in clustering by the relatedness of the ETs. ETs allowed the epidemiological characterization of stool isolates recovered from elderly inpatient residents and from adolescent chronic-care psychiatric patients. These results indicate that esterase electrophoretic typing may be a marker for epidemiological and ecological analyses.     

Studies on soybean trypsin inhibitors. X. Isolation and partial characterization of four soybean double-headed proteinase inhibitors

Four Bowman-Birk type double-headed inhibitors (B, C-II, D-II, and E-I) were isolated from soybeans. Inhibitor B was different from Bowman-Birk inhibitor only in chromatographic behavior. One mole of C-II inhibited one mole each of bovine trypsin and bovine alpha-chymotrypsin, probably at the same site, and porcine elastase at another reactive site. In the ordinary assay system D-II and E-I inhibited only trypsin activity at a non-stoichiometric inhibitor-enzyme ratio of 1:1.4, and the complexes had rather high dissociation constants. These inhibitors were all inactive toward subtilisin BPN'.     

Fast recruitment of recurrent inhibition in the cat visual cortex

Neurons of the same column in L4 of the cat visual cortex are likely to share the same sensory input from the same region of the visual field. Using visually-guided patch clamp recordings we investigated the biophysical properties of the synapses of neighboring layer 4 neurons. We recorded synaptic connections between all types of excitatory and inhibitory neurons in L4. The E-E, E-I, and I-E connections had moderate CVs and failure rates. However, E-I connections had larger amplitudes, faster rise-times, and shorter latencies. Identification of the sites of putative synaptic contacts together with compartmental simulations on 3D reconstructed cells, suggested that E-I synapses tended to be located on proximal dendritic branches, which would explain their larger EPSP amplitudes and faster kinetics. Excitatory and inhibitory synapses were located at the same distance on distal dendrites of excitatory neurons. We hypothesize that this co-localization and the fast recruitment of local inhibition provides an efficient means of modulating excitation in a precisely timed way.     

Quantitative structure-activity relationships for the pre-steady state of Pseudomonas species lipase inhibitions by p-nirophenyl-N-substituted carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1-6) are composed of two steps: (1) formation of the non-covalent enzyme-inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme-inhibitor adduct (E-I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, KS, and the rate constant for formation of the tetrahedral E-I adduct from the E:I complex, k2 are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k(obs)) versus inhibition concentration ([I]) plot against k(obs)=k2+k2[I]/(KS+[I]). Values of pKS, and log k2 are linearly correlated with the sigma* values with the rho* values of -2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the rho* value of -2.0. The tetrahedral E-I adducts on the other hand are more negative charges than the E:I complexes due to the rho* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.