Fatty Acid synthesis in endosperm of young castor bean seedlings

Enzyme assays on organelles isolated from the endosperm of germinating castor bean (Ricinus communis) by sucrose density gradient centrifugation showed that fatty acid synthesis from [¹⁴C]malonyl-CoA was localized exclusively in the plastids. The optimum pH was 7.7 and the products was mainly free palmitic and oleic acids. Both NADH and NADPH were required as reductants for maximum activity. Acetyl-CoA, and acyl-carrier protein from Escherichia coli increased the rate of fatty acid synthesis, while low O₂ levels suppressed synthesis. In the absence of NADPH or at low O₂ concentration, stearic acid became a major product at the expense of oleic acid. Fatty acid synthesis activity was highest during the first 3 days of germination, preceding the maximum development of mitochondria and glyoxysomes. It is proposed that the plastids are the source of fatty acids incorporated into the membranes of developing organelles.     

Characterization of lipase from an alkalophilic yeast sp.

Summary A lipase producing alkalophilic yeast species was isolated, identified, classified and termed as Candida BG-55, from a sample of nickel catalyst of a vegetable oils industry near Chandigarh, INDIA. The lipase from this microorganism had temperature and pH optima of 40°C and 8.5 respectively and was stable at 45°C for 4 hrs. Enzyme activity was stimulated by Ni⁺⁺ and Ca⁺⁺ ions whereas Fe⁺⁺ and Fe⁺⁺⁺ ions inhibited the activity.     

Effects of gibberellins, darkness and osmotica on endosperm rupture and class I β-1,3-glucanase induction in tobacco seed germination

The “Havana 425” cultivar of Nicotiana tabacum L. is photodormant. Gibberellins (e.g. 10^?5 M GA₄ or GA₇) can substitute for light in releasing dormancy. Measurements of β-1,3-glucanase activity, mRNA accumulation and the activity of the class I β-1,3-glucanase B promoter indicated that class I β-1,3-glucanases are induced by GA₄ in the dark in association with germination. As in the light, this induction occurred prior to endosperm rupture and was localized exclusively in the micropylar region of the endosperm where the radicle will penetrate. Abscisic acid (ABA, 10^?5 M) did not appreciably affect GA-induced release of photodormancy or seed-coat rupture, but it delayed endosperm rupture and inhibited the rate of class I β-1,3-glucanase accumulation. Seeds imbibed in the light in the presence of osmotica, e.g. 0.04 M polyethylene glycol 6000, showed delayed seed-coat and endosperm rupture, delayed onset of β-1,3-glucanase induction, and decreased rates of β-1,3-glucanase accumulation. These delays were shortened by GA₄ treatment. Our results suggest that GAs and ABA act at two distinct sites during germination and that expansive growth of the embryo acts in two ways by triggering β-1,3-glucanase induction and by providing force for endosperm penetration. This provides further support for our working hypothesis that class I β-1,3-glucanases promote endosperm weakening and facilitate radicle penetration.     

Studies on NADP-isocitrate dehydrogenase from maturing castor bean seeds

NADP-specific isocitrate dehydrogenase from the soluble fractionof maturing castor bean endosperm was partially purified (approximately180-fold) and some of its enzymatic properties were studied.Mg⁺⁺, Mn⁺⁺, Cd⁺⁺, Ba⁺⁺, Co⁺⁺, Zn⁺⁺, and Sr⁺⁺ were activatorsof the enzyme reaction at a concentration of 6.7x10 M. The optimumpH of this enzyme was about 8.5. The enzyme was stable in thenarrow range from pH 7.0 to pH 8.0. Km values for isocitrateand NADP at pH 8.5 were 3.5x10^–6 M and 3.6x10^–6M, respectively. Enzyme stability was not affected by NaCl concentrationand enzyme reaction was inhibited at 5x10^–6 M PCMB (80%inhibition). It is suggested that the condensation product ofglyoxylate and oxalacetate also inhibits the reaction. NADP-IDHin the crude extract from maturing castor bean endosperm washeat-stable but the dialyzed enzyme preparation and the partiallypurified enzyme were labile against heat treatment at 57°C.When Mg⁺⁺ was added to the partially purified enzyme in thepresence of isocitrate or NADP, the enzyme was stabilized againstheat treatment. Mn⁺⁺, Ca⁺⁺, Co⁺⁺, Sr⁺⁺ or Ba⁺⁺ could be substitutedfor Mg⁺⁺. Addition of only one of the factors, Mg⁺⁺, isocitrateor NADP, had no effect on the heat stability. Moreover, a combinationof isocitrate and NADP did not establish stabilization. A divalentcation plays a central role, while adenine nucleotide, especiallyATP, may have an important part in stabilization. (Received August 14, 1972; )     

Affinity Chromatographic Purification of β-Glucosidase of Candida Guilliermondii

Abstract

A 8-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethylcneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacryl-amide disc electrophoresis of the purified enzyme gave a single band. A K_m of 1.25 × 10^?4M was obtained using p_-nitrophenyl-β-D_-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at a temperature of 37°C. Enzyme activity was completely inhibited by Hg⁺⁺, Pb⁺⁺, and Zn⁺⁺ ions. The molecular weight of the enzyme is 48, 000 as estimated by sucrose density gradient centri-fugation.     

Amino acid metabolism in the pedicel-placenta-chalazal region of the developing maize kernel

The pedicel-placenta-chalazal (PPCh) region is a heterogeneous maternal tissue located at the base of the endosperm. Its active role in the transport and metabolism of nitrogen compounds supplied to the endosperm has been shown by incorporation of labelled compounds, as well as by analysis of appropriate enzyme activities and the free amino acid pool. Within 30 min incubation, 60–70% of the label given in [¹⁴C]aspartate or [¹⁴C]glutamate was recovered in the organic acid fraction of the PPCh. Although the dry matter and protein content of the PPCh region was practically unchanged between days 15 and 30 after pollination, glutamate transaminase, alanine transaminase, NADP-malic enzyme and NAD-malate dehydrogenase were increased substantially. The PPCh region contains glutamine at a significantly higher concentration and alanine at a lower concentration than the endosperm. In contrast to glutamate synthase, the activity of glutamine synthetase per endosperm and per mg protein is significantly higher in the PPCh region. This implies involvement of these tissues in the transport of nitrogen compounds, mainly in the form of glutamine, as well as possible compartmentation of the glutamine-glutamate cycle in the developing maize kernel.     

Induction, Purification and Characterization of α-1, 3-Glucanase from Brassica juncea L Infected with Albugo candida

Infection of Brassica juncea plants with Albugo candida caused the induction of β-1,3-glucanase in the leaf tissues following inoculation; the induction being more pronounced in the resistant cultivars. The enzyme purified from infected leaves of resistant cultivar RC-781 had a molecular mass of 35.4 kD. The enzyme exhibited its optimum activity at 50°C and at pH 5.5 with K_m value of 0.55 mg laminarin ml^?1. The enzyme activity was slightly stimulated by Cl^? and Mg⁺⁺, while inhibited by Hg⁺⁺ and Mn++ at higher concentrations only     

Effects of morphine tolerance and dependence on Mg++ dependent ATPase activity of synaptic vesicles.

The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg⁺⁺-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg⁺⁺-dependent ATPase and Na⁺, K⁺ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg⁺⁺-dependet ATPase was dependent on the concentration of Mg⁺⁺ but not of Ca⁺⁺; maximum activity was obtained with 2 mM MgCl₂. On the other hand, Mg⁺⁺-dependent ATPase activity of SPM was dependent on both Mg⁺⁺ and Ca⁺⁺, activity being maximum using 2 mM MgCl₂ and 10^?5 M CaCl₂. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.     

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.     

Biochemical characterization of a thermostable β-1,3-xylanase from the hyperthermophilic eubacterium, Thermotoga neapolitana strain DSM 4359

The biochemical properties of a putative β-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against β-1,3-xylan and released β-1,3-xylobiose and β-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3–10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble β-1,3-xylan K _m value was 10.35 mg/ml and the k _cat value was 588.24 s^?1. The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.     

Optimization and characterization of extracellular cellulase produced by Bacillus pumilus MGB05 isolated from midgut of muga silkworm (Antheraea assamensis Helfer)

Mature larvae of Antheraea assamensis were collected from different locations of Assam to isolate the cellulolytic gut microflora. Altogether sixty cellulase degrading bacteria were isolated on agar plates containing microcrystalline cellulose as the sole carbon source. Among them, ten isolates showed hydrolyzing zone on agar plates containing carboxy methyl cellulose (CMC) after staining with Congo-red. Isolate MGB05 exhibited the highest CMCase activity (0.262?U/mL) at 72?h of incubation under submerged condition. FPase and β-glucosidase activity were 0.012?U/mL and 3.71?U/mL respectively. It showed maximum FPase (0.022?U/mL) activity on the 3rd day of incubation in the media containing wheat bran as a carbon source. β-glucosidase production was also found to be highest with wheat bran (20.03?U/mL) at 48?h of incubation. The optimum pH and temperature of FPase activity of MGB05 were found at 6.0 and 50?°C respectively while for β-glucosidase activity, it was maximum at pH?6.0 under 50?°C. In addition, metal ion Mg⁺⁺ and Ca⁺⁺ enhanced FPase activity up to 110.92% (0.026?U/mL) and 105.31% (0.025?U/mL) respectively. In-vitro antimicrobial bioassay of the most potent cellulolytic bacteria (MGB05) also showed high antimicrobial activity against Escherichia coli (2.9?cm) and Pseudomonas aeruginosa (3.0?cm). The isolate MGB05 has been identified based on 16S rDNA homology as Bacillus pumilus MGB05 with accession KP298708.2. Results encompass the prospective beneficial role of gut-microflora on digestion and disease resistance, which might be a potential probiotic component to enhance silk productivity.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) was found not to inhibit active sodium transport across the isolated frog skin when added in 10^–3 m concentration to the basal-lateral surface. The same Cd⁺⁺ concentration similarly had no effect on Na⁺ transport across the isolated epithelial cell layer from the frog skin, although this dose of Cd⁺⁺ did inhibit Na⁺ transport across the frog urinary bladder and large intestine. When 10^–3 m Cd⁺⁺ was added to the apical surface of the isolated frog skin or to the isolated epithelial cells from the frog skin, sodium transport was reversibly stimulated, in contrast to the irreversible inhibition noted above. If equimolar cysteine was added with Cd⁺⁺ to the apical surface of the skin, however, active Na⁺ transport was irreversibly inhibited. In conjunction with the inhibition produced by equimolar Cd⁺⁺ and cysteine, isotopic Cd⁺⁺ permeation into the tissue was three times higher when added with cysteine than in the absence of cysteine. Thus, the effects of Cd⁺⁺ on epithelial Na⁺ transport is variable according to the epithelium studied and the presence of potential carrier molecules.     

Effects of beta-bungarotoxin on calcium uptake by sarcoplasmic reticulum from rabbit skeletal muscle

A fluorescent chelate probe and a Millipore filtration technique have been used to study the effects of β-bungarotoxin (β-toxin) on passive and active Ca⁺⁺ uptake and ATPase in fragmented sarcoplasmic reticulum (SR) of rabbit skeletal muscle. β-Toxin at 3 × 10^?6 M did not affect ATPase activity. In the absence of ATP, β-Toxin increased the passive uptake of Ca⁺⁺; in the presence of ATP, active Ca⁺⁺ uptake was inhibited. The effect of β-toxin in SR can be detected at concentrations as low as 10^?9 M. The results suggest that β-toxin induces Ca⁺⁺ leakage in SR membranes.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

Analysis of a calcium ion binding system composed of two different sites

Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca⁺⁺ binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca⁺⁺ binding sites and without such solute were titrated with Ca⁺⁺ using Mx as an indicator of free Ca⁺⁺ concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(β-aminoethyl)-N-N′-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca⁺⁺; the association constants of the high affinity site and low affinity site are 4.3 × 10⁵ M^-1 and 9 × 10³ M^-1, respectively. The advantages and limitations of this methodology are discussed.     

Appearance of sedoheptulose 1,7-diphosphatase activity on conversion of chloroplast fructose 1,6-diphosphatase from dimer form to monomer form.

Chloroplast fructose 1,6-diphosphatase isolated at pH 5.5 as the dimer dissociated to the monomer at pH 8.5. When the pH was adjusted from 8.5 back to 5.5, the newly formed monomer partly reassociated to form the dimer. The monomer lacked the fructose diphosphatase activity characteristic of the dimer (measured in the presence of a saturating concentration of Mg⁺⁺) but retained ferredoxin-dependent activity (measured in the presence of Mg⁺⁺ plus protein factor and either reduced ferredoxin or dithiothreitol). In addition, the monomer acquired sedoheptulose 1,7-diphosphatase activity that was dependent on either reduced ferredoxin or dithiothreitol and the protein factor.     

Muscle Contraction during Hyperpolarizing Currents in the Crab

Isolated muscle fibers from the motor legs of the crab Trichodactilus dilocarcinus were submitted to strong hyperpolarizing currents of varied intensities which produced tension during the current pulse. Threshold for tension was obtained with intensities of about 0.2 x 10^–5 A, changing E_m to ca. –150 mV (starting from a resting potential ofca. –80 mV). At the closure of the anodic square pulse, a second phase of tension usually appeared superimposed upon the one obtained during hyperpolarization. The first phase of tension increased with the increase of Ca⁺⁺ concentration in the bath. Sr⁺⁺ produced the same type of mechanical output as Ca⁺⁺. When added to the normal Ca⁺⁺ concentration, Ba⁺⁺ and Mn⁺⁺ in low concentrations (up to 21.5 mM) also increased the tension of this phase, but at higher concentrations they blocked both phases while Mg⁺⁺ did not alter the tension. Of all the divalent cations employed, only Sr⁺⁺ is capable of developing tension as a substitute for Ca⁺⁺ in the external media. Procaine administered in a dosage (5 x 10^–3 W/V)which would suppress the contracture due to caffeine (10 mM), did not modify the tension developed during the hyperpolarization. The preceding data indicate that the Ca⁺⁺ required for tension during hyperpolarization comes from sites which would differ from those usually postulated for tension due to depolarization in the muscle fibers of other crustaceans (American crayfish). Furthermore, the external source of Ca⁺⁺ appears to be one mainly implicated in the induction of tension due to inward current pulses.     

Studies on the Proteolytic Enzymes of Thermophilic Streptomyces

Proteolytic enzymes derived from thermophilic streptomyces sp. strain 1689 were purified and some properties were studied. A 8-fold purification was obtained from the culture supernatant by ammonium sulfate fractionation, acetone precipitation, and chromatography on CM-Sephadex. Two proteinases of almost identical properties were fractionated on CM-Sephadex chromatography. The purified preparations appeared to be homogeneous on ultracentrifugation. The optimum pH for proteolytic activity on casein was found to be pH 10.6~10.8. The stability was considerably increased by the addition of Ca⁺⁺, and the proteinases exhibited d relatively high thermal stability. Enzyme activity was inhibited by oxidizing agents, PCMB, potato inhibitor, DFP, and heavy metal ions. Na⁺, K⁺, Mg⁺⁺, and Fe⁺⁺ showed an activating effect.