Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification,properties, and amino acid sequences

Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is atpH 4.55 for LA-1 and atpH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 å. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0×10⁹ M^?1 sec^?1 for LA-1 and 0.8 × 10⁹ M^?1 sec^?1 for LA-2 and that of K₂HPO₄ quenching is 1.6×10¹¹ M^?1 sec^?1 for LA-1 and 1.2×10¹¹M^?1 sec^?1 for LA-2. Analysis of the circular dichroic spectra yields 40%α-helix and 60%Β-turn for La-1 and 45%α-helix and 55%Β-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzymeinhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.     

Sequence and expression of the discoidin I gene family in Dictyostelium discoideum

We have isolated recombinant phage and plasmids containing the four developmentally regulated discoidin I genes of Dictyostelium discoideum. Two of the genes are linked within 0.5 × 10³ bases with the same polarity. S¹ nuclease mapping shows that at least three members of the gene family are expressed and that the 5′ ends of the mRNAs start at equivalent sites. The genes have homologous 5′ untranslated regions and extremely divergent 3′ untranslated regions. In addition, some of the genes are flanked by homologous repeat sequences. The genes encode three different isoelectric forms of the protein. Examination of nucleotide sequences in the protein coding region shows that most nucleotide changes in the 5′ half of the gene result in amino acid substitutions while most base substitutions in the 3′ half are neutral.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

Sequence analysis of the murine Hox-2.2, -2.3, and -2.4 homeo boxes: evolutionary and structural comparisons

We have determined the nucleotide sequences and deduced the amino acid sequences of three tandemly arranged murine boxes of the Hox-2 homeo box gene complex on mouse chromosome 11 (Hox-2.2, -2.3, and -2.4). The type and position of differences with other sequenced homeo boxes were analyzed. Hox-2.2 is nearly identical with its cognate human homeo box Hu-2. Hox-2.3 shares 59 of 61 amino acids with the Antennapedia homeo domain of Drosophila and the MM-3 homeo domain of Xenopus and shows 60 of 61 amino acid identity with human HuC1. Hox-2.3, MM-3, and HuC1 also share a stretch of six glutamic acid residues followed by a stop codon 15-20 amino acids 3' of the homeo domain. Hox-2.4 is relatively divergent from most of the other homeo boxes sequenced to date; however, it matches the Hox-3.1 murine homeo domain at 60 of 61 positions. Sequence comparisons with other murine homeo domains, together with previous studies of their genomic organization and chromosomal location, provide support for the hypothesis of a large-scale duplication resulting in the two major murine homeo box gene complexes Hox-1 and Hox-2.     

A paucity of palindromes in φX174

The nucleotide sequence of the bacteriophage φX174 contains surprisingly few restriction endonuclease recognition sites. The observed frequency of those sites which consist of a six-nucleotide palindromic sequence would occur by chance with a probability of less than 8 × 10^?5. The genome of φX174 does not contain the four-nucleotide palindromic recognition site for the enzyme MboI and this finding has a probability of only 1·7 × 10^?9. A further analysis of the nucleotide sequence revealed that there is a marked scarcity of palindromic sequences with a length of four or six nucleotides while all other palindromes occur with a frequency close to that dictated by chance. A preliminary analysis of the genomes of other DNA viruses indicated that this palindrome avoidance is associated with single-stranded viruses only. The reasons for the paucity of these short even-numbered palindromes remains to be determined.     

Kinetics of phosphate uptake in excised maize root

The short term uptake of phosphate involving 10 min absorption followed by 5 min desorption, both at 30 °C, in the concentration range 1.0×10^?9 to 7.5×10^?2 M KH₂PO₄ by fresh and washed maize (Zea mays L. cv. Ganga Safed-2) roots can be described by a single isotherm having five phases (0 and I–IV) with regularly spaced kinetic constants. Almost identical kinetics were observed in both fresh and washed maize roots. The kinetics of phase 0 in the concentration range 1.0×10^?9–3.0×10^?5 M. was sigmoidal in fresh maize roots, however, in washed tissue exhibited 2 phases termed here as 0a and 0b. 0a covered the concentration range 1.0×10^?9–5.0×10^?6 M and 0b 6.0×10^?6–3.0×10^?5 M. In the concentration range 1.0×10^?4–7.5×10^?2 M four distinct phases, termed as I, II, III and IV were evident in both fresh and washed maize roots. Each phase obeyed Michaelis—Menten kinetics. The values of K_m and V_max have been estimated for each phase. The uptake isotherm was accompanied by discontinuous transitions.     

Suggestion of GLYAT gene underlying variation of bone size and body lean mass as revealed by a bivariate genome-wide association study

Bone and muscle, two major tissue types of musculoskeletal system, have strong genetic determination. Abnormality in bone and/or muscle may cause musculoskeletal diseases such as osteoporosis and sarcopenia. Bone size phenotypes (BSPs), such as hip bone size (HBS), appendicular bone size (ABS), are genetically correlated with body lean mass (mainly muscle mass). However, the specific genes shared by these phenotypes are largely unknown. In this study, we aimed to identify the specific genes with pleiotropic effects on BSPs and appendicular lean mass (ALM). We performed a bivariate genome-wide association study (GWAS) by analyzing ~690,000 SNPs in 1,627 unrelated Han Chinese adults (802 males and 825 females) followed by a replication study in 2,286 unrelated US Caucasians (558 males and 1,728 females). We identified 14 interesting single nucleotide polymorphisms (SNPs) that may contribute to variation of both BSPs and ALM, with p values <10^?6 in discovery stage. Among them, the association of three SNPs (rs2507838, rs7116722, and rs11826261) in/near GLYAT (glycine-N-acyltransferase) gene was replicated in US Caucasians, with p values ranging from 1.89 × 10^?3 to 3.71 × 10^?4 for ALM–ABS, from 5.14 × 10^?3 to 1.11 × 10^?2 for ALM–HBS, respectively. Meta-analyses yielded stronger association signals for rs2507838, rs7116722, and rs11826261, with pooled p values of 1.68 × 10^?8, 7.94 × 10^?8, 6.80 × 10^?8 for ALB–ABS and 1.22 × 10^?4, 9.85 × 10^?5, 3.96 × 10^?4 for ALM–HBS, respectively. Haplotype allele ATA based on these three SNPs was also associated with ALM–HBS and ALM–ABS in both discovery and replication samples. Interestingly, GLYAT was previously found to be essential to glucose metabolism and energy metabolism, suggesting the gene’s dual role in both bone development and muscle growth. Our findings, together with the prior biological evidence, suggest the importance of GLYAT gene in co-regulation of bone phenotypes and body lean mass.     

Breeding and characterization of amino acid-analogue-resistant mutants of Arthrospira platensis

To obtain amino acid-analogue-resistant mutants the wild strain A9 of Arthrospira platensis was mutated by ethylmethane sulfonate (EMS). Mutagenic effects of strain A9 by EMS were studied. The experimental results indicated that the survival rate curve of strain A9 took a typical “exponential shape” with lethal dosage of EMS being 1 %. The survival of A9 strain was 13.2 % when treated with 0.4 % of EMS, and the resistant mutation rates to two amino acid analogues, ρ-fluorophenylalanine (FPA) and L-canavanine sulphate (CS), were greatly increased with the highest rates being at 4.9 × 10^?4 and 3.24 × 10^?4, respectively. By repeated screening, two stable mutants resistant to amino acid analogues, A9f resistant to FPA and A9c resistant to CS, were obtained. Resistances of the two mutants to corresponding amino acid-analogues were both significantly increased. Compared with their parent strain A9, A9f appeared larger than A9 performance in filament diameter, spiral diameter, spiral pitch, filament length and spiral number, and A9c showed much longer length and spiral pitch than those of the initial strain. Analysis results on amino acids compositions and contents showed that both two mutants accumulated quite higher concentration of amino acids in cells. The two mutants might be excellent high amino acids producing strain. By this means two useful mutants with stable genetic makers for further genetic study of A. platensis were obtained, which laid a good foundation for further study on the transformation of A. platensis.     

Interaction of Kinetin and Abscisic Acid in the Avena Straight-Growth Test

Different amounts of abscisic acid, 2.7 × 10^?9? 5 × 10^?8 moles, were chromatographed in isopropanol: ammonia: water (100:14:6), firstly alone and secondly together with 5 × 10^?8 moles kinetin. The same amount of kinetin was also chromatographed alone. The chromatograms were tested biologically with the Avena straight-growth test. Whereas a large part of the chromatograms of kinetin gives growth stimulation, the Rf region 0.4–0.6 of abscisic acid chromatograms is strongly growth-inhibiting. The inhibition within this Rf region does not become less if abscisic acid and kinetin are chromatographed together.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.     

pH effects on growth and lipid accumulation of the biofuel microalgae Nannochloropsis salina and invading organisms

Biofuels derived from non-crop sources, such as microalgae, offer their own advantages and limitations. Despite high growth rates and lipid accumulation, microalgae cultivation still requires more energy than it produces. Furthermore, invading organisms can lower efficiency of algae production. Simple environmental changes might be able to increase algae productivity while minimizing undesired organisms like competitive algae or predatory algae grazers. Microalgae are susceptible to pH changes. In many production systems, pH is kept below 8 by CO₂ addition. Here, we uncouple the effects of pH and CO₂ input, by using chemical pH buffers and investigate how pH influences Nannochloropsis salina growth and lipid accumulation as well as invading organisms. We used a wide range of pH levels (5, 6, 7, 8, 9, and 10). N. salina showed highest growth rates at pH 8 and 9 (0.19?±?0.008 and 0.19?±?0.011, respectively; mean ± SD). Maximum cell densities in these treatments were reached around 21 days into the experiment (95.6?×?10⁶?±?9?×?10⁶ cells mL^?1 for pH 8 and 92.8?×?10⁶?±?24?×?10⁶ cells mL^?1 for pH 9). Lipid accumulation of unbuffered controls were 21.8?±?5.8 % fatty acid methyl esters content by mass, and we were unable to trigger additional significant lipid accumulation by manipulating pH levels at the beginning of stationary phase. Ciliates (grazing predators) occurred in significant higher densities at pH 6 (56.9?±?39.6?×?10⁴ organisms mL^?1) than higher pH treatments (0.1–6.8?×?10⁴ organisms mL^?1). Furthermore, the addition of buffers themselves seemed to negatively impact diatoms (algal competitors). They were more abundant in an unbuffered control (12.7?±?5.1?×?10⁴ organisms mL^?1) than any of the pH treatments (3.6–4.7?×?10⁴ organisms mL^?1). In general, pH values of 8 to 9 might be most conducive to increasing algae production and minimizing invading organisms. CO₂ addition seems more valuable to algae as an inorganic carbon source and not as an essential mechanism to reduce pH.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration

The response of the three major classes of puff in salivary gland chromosomes of larval Drosophila melanogaster to varying β-ecdysone concentrations has been studied in in vitro cultured glands. Two (25AC and 68C) of the intermolt puffs regress at a rate dependent upon the hormone concentration. Three rapidly reacting puffs (23E, 74EF and 75B) respond in a graded way to β-ecdysone concentrations over a range of at least 600 ×. In contrast, five late-reacting puffs (62E, 78D, 22C, 63E, and 82F) do not respond below 5 × 10^?8M and at 2.5 × 10^?7M react maximally. The 50% response of the early puff sites 74EF and 75B and of the late puff sites occurs at 1 × 10^?7M. Two points are discussed in detail: whether ecdysone is necessary as a sustained stimulus or only as a trigger for the sequential puffing response and an evaluation of the absolute ecdysone concentration necessary for induction.     

A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids

Leukotriene A₄ hydrolase (LTA4H––EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA₄ through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate (k _cat/K _m values). Among them, the benzyl ester of aspartic acid exhibited a k _cat/K _m value that was more than two orders of magnitude higher (1.75 × 10⁵ M^?1 s^?1) as compared to l-Arg (1.5 × 10³ M^?1 s^?1). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H.     

Rabbit brain purine nucleoside phosphorylase. Physical and chemical properties. Inhibition studies with aminopterin, folic acid and structurally related compounds.

Rabbit brain purine nucleoside phosphorylase used in this study was purified 6000-fold to apparent homogeneity and a specific activity or 50 μmol min^?1 mg ^?1 protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound d,l-6-methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 × 10^?5M for folic acid and 3.80 × 10^?5m for d,l-6-methyl 5,6,7,8-tetrahydropterine. Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 × 10^?4 and 1.95 × 10^?4m. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory potency with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 × 10^?4m. The Michaelis constant for the substrate guanosine was near 6.0 × 10^?5m. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both histidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependent sensitivity of an essential catalytic group, and its probable location at the active site.     

Molecular cloning,sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^? mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene

Regions of the mouse and human genomes with strong homology to the Drosophila engrailed gene have been identified by Southern blot analysis. One mouse engrailed-like region, Mo-en.1, has been cloned and partially sequenced; homology with the engrailed gene is localized to a 180 bp engrailed-like homeo box and 63 nucleotides immediately 3' to it. The protein sequence this region can encode includes 81 amino acids, of which 60 (75%) are identical with those of the putative translation product of the corresponding engrailed sequence. These data suggest that Mo-en.1 represents a mouse homolog of a gene of the Drosophila engrailed gene complex. Mo-en.1 has been mapped to chromosome 1, indicating it is not linked to other homeo box sequences that have been mapped in the mouse genome. Analysis of poly(A)+ RNA extracted from teratocarcinoma cells and whole mouse embryos demonstrates that the conserved homeo box region of Mo-en.1 is expressed differentially during mouse embryogenesis.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.