Formation of 2-hexenal from linolenic acid by macerated Ginkgo leaves

The content of linolenic acid and its fat-soluble derivatives in Ginkgo leaves has been determined. By utilization of uniformly ¹⁴C-labelled linolenic acid it has been shown that the linolenic acid in Ginkgo leaves is converted into 2-hexenal when the leaves are macerated in the presence of air. The conversion of linolenic acid to 2-hexenal under the conditions of temperature and pH existing in the Ginkgo leaf requires the presence in the leaves of an enzyme or other catalyst. This is not lipoxidase but is a hexane-insoluble, water-soluble substance. A preparation of this substance strongly catalyzes the absorption of oxygen by linolenic acid in water at 20°.     

Enzymic isoprenylation of tryptophanyl cyclic dipeptides by Aspergillus amstelodami

Cyclo-l-prolyl-l-tryptophanyl, a component of several metabolites of Aspergillus ustus and cyclo-l-alanyl-l-tryptophanyl were compared as co-substrates with 3-methyl-2-butenyl-[1-³H]-1-pyrophosphate for an enzyme from A. amstelodami which previously had been described to isoprenylate cyclo-l-alanyl-l-tryptophanyl. Both compounds were equally active as isoprene acceptors using the A. amstelodami enzyme. The mass spectrum of the isoprenylated cyclo-l-prolyl-l-tryptophanyl indicated that the product was a monoisoprenylated derivative of the starting cyclic dipeptide. The most probable structure for this enzymic product is cyclo-l-prolyl-2(1,1-dimethylallyl)-l-tryptophanyl. The cyclo-pentylidene analogue of 3-methyl-2-butenyl-1-pyrophosphate did not serve as an alkylating agent when cyclo-l-alanyl-l-tryptophanyl was used as co-substrate.     

Mono- and diphenolase activity from fruit of Malus pumila

Apple fruit used for beverage production has a polyphenol oxidase which does not hydroxylate tyrosine under any conditions but it hydroxylates p-coumaric acid in the presence of NADH, and phloridzin in the absence of cofactors. The apparent K_ms for hydroxylation of phloridzin and p-coumaric acid are 1.5 and 4 mM, respectively. However, subsequent oxidation of 3-hydroxyphloridzin or caffeic acid has an apparent K_m of 200 nM. The oxidation products of 3-hydroxyphloridzin are complex and a stable dimeric quinone is finally formed. The apparent K_ms for oxidation of catechin, epicatechin, chlorogenic acid, l-Dopa and 4-methyl catechol are 4.7, 5.7, 6.0, 2.7 and 3.2 mM, respectively. The K_m for oxygen was 4.3 % although there was marked substrate inhibition by oxygen above 30 %. Polyphenol oxidase was stable at pH 3.5–4.5 with an optimum of 4.5.     

Extent of copper LDL oxidation depends on oxidation time and copper/LDL ratio: chemical characterization

The aim of our study was to determine, as a function of [Cu(2+)]/[LDL] ratios (0.5 and 0.05) and of oxidation phases, the extent of LDL oxidation by assessing the lipid and apo B oxidation products. The main results showed that: (i) kinetics of conjugated diene formation presented four phases for Cu(2+)/LDL ratio of 0.5 and two phases for [Cu(2+)]/[LDL] ratio of 0.05; (ii) oxidation product formation (cholesteryl ester and phosphatidylcholine hydroperoxides, apo B carbonyl groups) occurred early in the presence of endogenous antioxidants, under both copper oxidation conditions; (iii) apo B carbonylated fragments appeared when antioxidants were totally consumed at [Cu(2+)]/[LDL] ratio of 0.5; and (iv) antioxidant concentrations were stable, oxysterol formation was negligible, and no carbonylated fragment was detected at [Cu(2+)]/[LDL] ratio of 0.05. Depending on the copper/LDL ratio, oxidized LDL differ greatly in the nature of lipid peroxidation product and the degree of apo B fragmentation.     

Middle Jurassic Ginkgo leaves from the Daohugou area,Inner Mongolia,China and their implication for palaeo-CO2 reconstruction

Well-preserved Ginkgo leaves with cuticle were collected from the Middle Jurassic Jiulongshan Formation in the Daohugou area, Inner Mongolia, China, which form the common elements in the Daohugou flora. Three new species of Ginkgo are recognized and their leaf morphology and cuticular structure are described for the first time. Two different pCO₂ proxy models were applied to reconstruct palaeo-atmospheric CO₂ concentration by using these new Ginkgo material. NLE (nearest living equivalent) suggests a semi-quantitative pCO₂ estimate as 839 ± 99 ppmv with Carboniferous standardization and 419 ± 49 ppmv with Recent standardization. While Barclay’s revised SI-pCO₂ regression suggests the estimates as 405 ± 71 ppmv. The results show that the pCO₂ estimate is higher than today’s atmospheric CO₂ concentration but lower than most of the other results on the Jurassic Period. Combined with the floristic composition, the climate at 165 Ma in the Daohugou area is inferred to be warm temperate.     

Enzymic formation of carbonyls from linoleic acid in leaves of Phaseolus vulgaris

Homogenization of Phaseolus vulgaris leaves at acid pH results in the evolution of hexanal, cis-3- and trans-2-hexenal. With cell-free extracts of leaves, linoleic and linolenic acids are enzymically converted to their hydroperoxides (predominantly the 13-hydroperoxide isomers) and to hexanal or hexenal respectively. Activity was highest in young, dark-green leaves and was stimulated by Triton X-100. Oleic acid is not a substrate for these reactions. Both 9- and 13-hydroperoxides were cleaved to carbonyl fragments and are proposed as intermediates in the formation of volatile aldehydes and non-volatile ω-oxoacids in P. vulgaris leaves. Properties of the enzyme systems are described.     

Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum

The aim of this work was to establish the pathways of carbohydrate oxidation used in the dark by leaves of Pisum sativum and Triticum aestivum. Segments of young and mature leaves of pea released the carbons of glucose-[¹⁴C] as ¹⁴CO₂ in the order 3,4 > 1 > 2 > 6 whereas in segments of young and mature leaves of wheat the order was 3,4 > 1 > 6 > 2. The detailed labelling of the constituents of mature leaves of wheat by glucose-[1-¹⁴C], -[2-¹⁴C], -[3,4-¹⁴C], and -[6-¹⁴C] was determined and showed that the high yield of CO₂ from C-6 relative to that from C-2 was due to release of C-6 during pentan synthesis. Estimates were made of the maximum catalytic activities of phosphofructokinase and glucose-6-phosphate dehydrogenase in pea and wheat leaves of three ages. The results of all the above investigations strongly indicate that both pea and wheat leaves in the dark oxidize carbohydrate via glycolysis and the pentose phosphate pathway with the latter accounting for no more than a third of the total. No evidence was obtained of any major change in the relative activities of the two pathways during the development of either type of leaf.     

Enzymic synthesis of corynanthe-type alkaloids in cell cultures of Catharanthus roseus: Quantitation by radioimmunoassay

The soluble enzyme system from Catharanthus roseus cell suspension cultures which synthesizes ajmalicine, 19-epiajmalicine and tetrahydroalstonine from tryptamine and secologanin has been further characterized. The enzymic reaction was followed quantitatively by using a radioimmunoassay (RIA) with antibodies directed against ajmalicine. This RIA proved exceedingly useful in determining the enzymology of the reaction and displayed a sensitivity shown previously only by radio-tracer methods. By this method, the enzyme system was found to function optimally at pH 6.5. Sensitivity of the enzymic synthesis of ajmalicine and its stereoisomers to δ-d-gluconolactone, a β-glucosidase inhibitor, indicated the involvement of a β-glucosidase in the formation of these alkaloids. The enzyme system catalysed the formation of unnatural ajmalicine analogues from ring-substituted tryptamines.     

Occurrence of an oxalate oxidase in Sorghum leaves

An oxalate oxidase found in the 15 000 g supernatant of 10-day-old sorghum leaves exhibited a pH optimum of 5 and a temperature optimum of 45° and was unaffected by Na⁺. The enzyme activity remained linear up to 10 min and the apparent K_m for oxalate was 2.4 × 10^?5 M. The enzyme activity was strongly inhibited by sodium dithionite and α,α′-dipyridyl. Inhibition by the latter was specifically reversed by Fe²⁺. The activity of the dialysed enzyme was restored by the addition of Fe²⁺ and FAD. Inhibition of the enzyme by iodoacetate, p-chloromercuribenzoate and N-methylmaleimide revealed that SH groups at the active site are essential.     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

酶解法提取纯化虎杖提取物中自藜芦醇的工艺研究

本文对虎杖提取物中虎杖苷的酶解条件及苷元白藜芦醇的提取纯化工艺进行研究,以样品中自藜芦醇的含量为指标,对纤维素酶、β-葡萄糖苷酶、复合酶进行筛选,结果表明以复合酶的水解效率最高:采用正交实验对影响复合酶酶解的因素:加酶量、温度、酶解时间进行考察;并对酶解后提取物中自藜芦醇的提取纯化工艺进行研究.得出如下较理想的酶解条件和提取纯化工艺:虎杖提取物,加水(pH 5)10倍,加20%的复合酶,于50℃保温24 h;酶解后的提取物经水、乙醇-水、碱溶液分步溶解沉淀,得白藜芦醇粗品,含量可达65%,工艺稳定可行.     

Fractionation by velocity sedimentation of Torpedo nicotinic post-synaptic membranes

Velocity sedimentation on sucrose gradients containing Torpedo physiological saline has been utilized to fractionate Torpedo (Torpedo californica and T. nobiliana) post-synaptic membranes isolated initially on the basis of their density by equilibrium centrifugation. Membranes are separated into two populations: (1) those retained within the gradient (referred to as gradient pool); and (2) membranes sedimenting rapidly through the gradient (referred to as f 22, fraction 22 of the gradient). Comparison of their polypeptide compositions by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicates that the gradient pool consists of highly purified nicotinic post-synaptic membranes containing the peptides of the acetylcholine receptor and a peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000, while f 22 contains the contaminating membranes present in the initial suspension as well as a small fraction of the nicotinic post-synaptic membranes. On the basis of the kinetics of efflux of ²²Na⁺ from the membrane fractions, it is concluded that the gradient pool contains most of the sealed vesicles with functional nicotinic receptors. The internal volume (μl/mg protein) of those membranes exceeds that of f 22 by a factor of 4, and greater than 85% of that internal volume is equilibrated by the nicotinic agonist carbamylcholine, while for f 22 only 40% is equilibrated. Thin-section electron microscopy has been used to estimate the distribution of vesicle sizes. The observed distribution for the gradient pool indicates that these vesicles are a size homogeneous population of diameter 0.3 μm, while f 22 contains a number of smaller and larger vesicles. Torpedo post-synaptic membranes have been treated with alkali to remove the non-receptor peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000. After alkaline extraction, velocity sedimentation permits the isolation of a population of size-homogeneous and well-sealed vesicles containing only the peptides of the nicotinic receptor. It is concluded that upon homogenization, the innervated surface of the Torpedo electroplax tends to form vesicles of uniform size (0.3 μm) which can be readily isolated by velocity sedimentation and that the peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000 is not required for the maintenance of bilayer structure.     

Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli

Recently, the smallest bioluminescent protein (MW: 19.9 kDa), Gaussia luciferase (GLuc), has been isolated from the marine copepod Gaussia princeps and has attracted much attention as a reporter protein. However, preparation of large quantities of homogeneous natively folded recombinant GLuc appears to be difficult due to its ten cysteines. Here, we report the biophysical characterization of recombinant GLuc expressed using a novel Escherichia coli expression system based on a cold induced expression vector (pCold). Using this system, a large fraction of the protein was expressed in the soluble fraction. GLuc, purified exclusively from the supernatant using nickel affinity chromatography, yielded a large amount of pure GLuc with a native disulfide bond pattern (Soluble-GLuc). Soluble-GLuc had a strong bioluminescence activity and it retained 65% of its activity after 30 min incubation at 95 °C. Soluble-GLuc remained fully folded until 40 °C, as assessed by circular dichroism; and the thermal denaturation curve was S-shaped, indicating a cooperative transition, with a midpoint temperature of 56 °C. These results indicate that both the structure and bioluminescence activity of GLuc remain stable at high temperatures, and they strongly suggest GLuc's potential as a reporter protein.     

Properties of two apyrases from Solanum tuberosum

Two homogeneous isoenzymes of apyrase from Pimpernel and Desirée varieties of Solanum tuberosum were obtained by affinity chromatography on agarose-Cibacron Blue or agarose-ATP-phosphonate columns. Both enzymes split POP bonds of organic and inorganic di- and triphosphates. The ratio of ATPase/ADPase is different for the two apyrases: 10 for Pimpernel and 1 for Desirée. All these activities require bivalent metals. Both isoapyrases have the same MW (49 000) but differ in their pI (8.74 for Pimpernel and 6.69 for Desirée). The optimum pH of hydrolysis of organic di- and triphosphates is 6 (except for Pimpernel ADPase) and 5 for inorganic substrates. Chemical modification of tryptophan, tyrosine, arginine and carboxylic residues decreased all enzymic activities of both enzymes. Protection by substrates and inactivation rates of the individual activities are different for each isoenzyme.     

Dammarane saponins of leaves of Panax pseudo-ginseng subsp. himalaicus

From dried leaves of Panax pseudo-ginseng subsp. himalaicus collected in Eastern Himalaya, new dammarane saponins, named pseudo-ginsenosides-F₁₁ and -F₈ were isolated along with the known Ginseng-root saponins, ginsenosides-Rb₃, Rd and -Re. Pseudo-ginsenoside-F₈ was proved to be a mono-acetyl-ginsenoside-Rb₃ and the location of its acetyl group was established mainly by ¹³C NMR spectroscopy. Pseudo-ginsenoside-F₁₁, was identified as the 6-O-α-rhamnopyransyl(1 → 2)-β-glucopyranoside of 3β,6α,12β,25-tetrahydoxy-(20S,24R)-epoxy-dammarane. The C-24 configuration of ocotillone and its related triterpenes was confirmed to be 24R excluding the recent comment by Lavie et al.     

Enzymic reduction of codeinone in vitro cell-free systems from Papaver somniferum and P. Bracteatum

Cell-free extracts have been prepared from Papaver somniferum which catalyze the reduction of codeinone-[16-³H] to codeine. The methodology for examining this conversion has pointed to conditions for exploring the preparation of suitable enzyme extracts. P. bracteatum also yielded a cell-free system which reduced codeinone to codeine, both of which are foreign to this species.     

Polyamine oxidation by enzymes from Hordeum vulgare and Pisum sativum seedlings

The properties of the amine oxidases of barley leaves and pea seedling cotyledons have been compared using a colorimetric assay in which the hydrogen p