几种担子菌胞外漆酶同工酶谱的研究

从猴头(Hericium erinaceus)、亚侧耳(Hohenbuehelia serotina)、毛柄金钱菌(Co-llybia velutipes)、多脂鳞伞(Pholiota adiposa)、香菇(Lentinus edodes)、黑木耳(Auriculariaauricula)、草菇(Volvaria volpacea)二孢蘑菇(Agaricus bisporus)和紫孢侧耳(Pleurotussapidus)、侧耳(Pleurotus ostreatus)风尾菇(P.sajor-caju)、金顶侧耳(P.citrinopileatus)、鲍鱼菇(P.cystidiosus)、佛罗里达侧耳(P.florida)、爪哇漏斗状侧耳(P.sajor-caju var.javanicus)商品化菌种的培养滤液里所得到的胞外漆酶(LC),采用聚丙烯酰胺凝胶等电聚焦盘状电泳方法,分析比较了它们的胞外漆酶同工酶。经多批次比较试验,获得了侧耳属七个种和八个不同属间各自恒定的漆酶同工酶谱。在这些酶谱中,分别包含了 A 组(pH3—5)和 B 组(pH5—8)的谱带。而且,所有菌种间漆酶带的等电点值(pI)各有异同。没有测定菌种间各漆酶带含量差别。讨论了漆酶同工酶谱用于分类研究的可能性。     

Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα-, αγ-, and γγ-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα-enolase was the predominant isoenzyme, and αγ-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα-enolase and a lower proportion of γγ-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα-enolase and lowest proportion of γγ-enolase.     

Higher-plant chloroplast and cytosolic fructose-1,6-bisphophosphatase isoenzymes: origins via duplication rather than prokaryote-eukaryote divergence

Full-size cDNAs encoding the precursors of chloroplast fructose-1,6-bisphosphatase (FBP), sedoheptulose-1,7-bisphosphatase (SBP), and the small subunit of Rubisco (RbcS) from spinach were cloned. These cDNAs complete the set of homologous probes for all nuclear-encoded enzymes of the Calvin cycle from spinach (Spinacia oleracea L.). FBP enzymes not only of higher plants but also of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a eubacterial origin of these eukaryotic nuclear genes. Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution. Both FBP and SBP of higher plant chloroplasts have acquired substrate specificity, i.e. have undergone functional specialization since their divergence from bifunctional FBP/SBP enzymes of free-living eubacteria.Abbreviations FBP fructose-1,6-bisphosphatase - SBP sedoheptulose-1,7-bisphosphatase - FBA fructose-1,6-bisphosphate aldolase     

Evolutionary analysis of aspartate aminotransferases

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family. Correspondence to: C.J. Marshall     

Wheat ribosome-inactivating proteins: Seed and leaf forms with different specificities and cofactor requirements

Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain VII which has previously been identified as the site of action of ricin A-chain. Tritin-S and tritin-L differ in both their ribosome substrate specificities and cofactor requirements. Tritin-S shows only barely detectable activity on ribosomes from the endosperm, its tissue of synthesis, whereas tritin-L is highly active on leaf ribosomes. Additionally, tritin-S is inactive on wheat germ, tobacco leaf and Escherichia coli ribosomes but active on rabbit reticulocyte and yeast ribosomes. Tritin-L is active on ribosomes from all of the above sources. Tritin-S, unlike tritin-L shows a marked requirement for ATP in its action.Abbreviations CM carboxymethyl - FPLC fast protein liquid chromatography - NEPHGE non-equilibrium pH gradient gel electrophoresis - PAP pokeweed antiviral protein - RIP ribosome-inactivating protein A.J.M. was the recipient of a U.K. Science and Engineering Research Council CASE studentship sponsored by Agricultural Genetics Company Ltd., Cambridge CB4 4GG, UK.     

Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca²⁺ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.Abbreviations DAB diaminobenzidine - GTA equal proportions of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol - TE tracheary element The authors are very grateful to Professor M. Tanahashi of Gifu University for providing hydroxycinnamyl alcohols. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to H.F.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

Tissue Specific Expression of Glutathione S-transferases, Glutathione Content and Lipid Peroxidation in Camel Tissues

Differential expression of glutathione S-transferase (GST) enzyme activity in various tissues of the camel was observed with a maximum activity in the liver. Compared with the rat and human livers, GST activity in camel liver was 50% lower than that of rat liver and similar to that of human liver. Extrahepatic tissues in camel have a comparable GST activity with those of similar tissues in the rat. Assay of GST activity using ethacrynic acid as substrate demonstrated maximum activity in the camel brain followed by intestine, liver and kidney. Microsomal GST activity in camel tissues was expressed in the order of liver > testis > intestine ≈ kidney ≈ brain. Phenotyping of GST was performed in camel hepatic and extrahepatic tissues using human specific antibodies to class α, μ, and π cytosolic GST isoenzymes and rat specific antibody to the microsomal GST. Western immunoblot and immunohistochemical analyses showed an abundant expression of GST α and μ in the camel liver, while π was very poorly expressed. Camel extrahepatic tissues however, had a significant expression of GST π. The camel GST isoenzymes were found to be predominantly expressed in the hepatocytes around the central vein with a gradual decrease in expression in the hepatocytes located toward the periphery. Kidney cortex exhibited a greater expression of the enzyme protein in the proximal tubules as compared to the glomeruli. Glutathione (GSH) concentration in rat tissues, except in the brain, was about 2-fold higher than that of camel tissues. Rate of NADPH-dependent microsomal lipid peroxidation was comparable both in the rat and camel tissues with the highest activity in the brain and lowest activity in the intestine. The differential expression of GST isoenzymes in different organs of the camel, GSH concentration and the rate of lipid peroxidation in different tissues may be important factors in determining the differential susceptibility of camel tissues to the toxic effects of xenobiotics.     

Isoforms of Glutamine Synthetase in the Marine Coccolithophorid Emiliania huxleyi (Prymnesiophyceae)

Two isoenzymes of glutamine synthetase (EC 6.3.1.2), GS₁ and GS₂, have been purified from cells of Emiliania huxleyi using Cibacron blue dye ligand chromatography and gel filtration, separated by ion-exchange chromatography on Mono-Q and partly characterized. Each enzyme is a homohexamer with a molecular mass of 402 kDa for GS₁ and 501 kDa for GS₂. The molecular mass of the subunits of GS₁ and GS₂ was estimated to be 61 and 78 kDa, respectively. As in higher plants, GS₁ is slightly more thermostable than GS₂ and much less stimulated by thiols than GS₂. For these reasons, GS₁ was designated as the cytosolic enzyme and GS₂ as the chloroplastic one. Although the K_ms for NH₂OH are about the same, GS₂ possesses a much higher affinity for glutamine than GS₁. As in bacteria, ATP appears to play an important role in the allosteric regulation of GS₂. l-Ala and CTP are potent inhibitors of GS₁ activity. CTP, carbamoyl-phosphate and l-Ala exert a cumulative inhibitory effect on GS₁ activity. GS₂ is also inhibited to some extent by l-Ala and l-His. NH₂-terminal sequence analysis of GS₂ did not show any homology with bacteria, cyanobacteria or higher plants.     

PP333对苹果幼树越冬期间过氧化物酶同工酶的影响

以越冬性强的元帅苹果幼树为对照,用PP333处理越冬性弱的金冠苹果幼树,测定越冬期间其枝条过氧化物酶同工酶的变化,并在翌年春季田间调查越冬情况。结果表明,PP333处理大大增加了过氧化物酶同工酶中与抗寒性有关酶的酶活,并提高了金冠苹果幼树的越冬性。