菜心中高等电点高活性的乙醇酸氧化酶同工酶的纯化和特性

乙醇酸氧化酶 (EC 1 1 3 15 ,GO)被认为只含4 0kD一种碱性亚基 ,是因为从多种植物中获得了具GO活性的蛋白 ,SDS PAGE后呈约 4 0kD单带[1] .菠菜GOcDNA编码约 370个氨基酸 ,即 4 0kD多肽 ,其碱 酸性氨基酸的比例高达 0 96 ,富含碱性氨基酸[2 ] .已克隆的GOcDNA在E .coli中表达     

Molecular basis for the isozymes of bovine glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase exists as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis, isoelectric focusing, and ion-exchange chromatography. GPI from bovine heart was purified to homogeneity and each of the isozymes resolved. Four of the five isozymes were characterized with regard to their physical, chemical, and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118,000) and subunit (59,000) molecular weights but had different apparent pI values of 7.2, 7.0, 6.8, and 6.6. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes in either reaction direction. Structural analyses showed that the amino termini were blocked and the carboxyl terminal sequences were -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes at pH extremes, at high ionic strength, in the presence of denaturants, or upon exposure to proteases. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release of ammonia along with the spontaneous shift to more acidic isozymes indicates deamidation as the molecular basis for the formation of the acidic isozymes both in vivo and in vitro. The change in the peptide fragmentation patterns following cleavage by hydroxylamine further suggests that deamidation of specific Asn-Gly bonds accounts for the structural basis of the isozymes.     

Direct detection of beta-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoelectrofocusing gels

A procedure to assay isozymes of beta-1,3-glucanase directly on polyacrylamide gel electrophoresis (PAGE) and isoelectrofocusing (IEF) gels by using 2,3,5-triphenyltetrazolium chloride is described. The reagent reacts with reducing sugars released by beta-1,3-glucanases from the substrate laminarin. Acidic and neutral isozymes of beta-1,3-glucanase were detected and quantified on 17.5% native PAGE gels run with an anodic buffer system. A significant linear relationship (alpha = less than 0.01, R = 0.991) was observed between amounts of beta-1,3-glucanase loaded and intensity of bands stained with the reagent on native PAGE gels. A full isozyme pattern was obtained on 7.5% IEF gels with a pH range of 3.5-9.5. The IEF gels were heated in a microwave oven during the staining process to minimize diffusion.     

The synthesis of a labile triosephosphate isomerase isozyme in human lymphoblasts and fibroblasts

Human peripheral lymphocytes contain a single electrophoretic form of triosephosphate isomerase (pI = 5.6). However, when induced to undergo blastogenesis by mitogens such as phytohemagglutinin or convanavalin A, a second isozyme (pI = 5.2) is also produced. This new isozyme is also found in human fibroblasts, but is present only in low concentrations in most other human tissues. The two isozymes were isolated from lymphocytes, lymphoblasts, and fibroblasts by isoelectric focusing, and their properties and the requirements for their synthesis were studied. The production of a new isozyme occurs concomitantly with blastogenesis and DNA synthesis, but when DNA synthesis is delayed by hydroxyurea, the appearance of the new isozyme is unaffected. The formation of the new isozyme is inhibited by actinomycin D and puromycin, and thus, appears to be dependent on both RNA and protein synthesis. Lymphocytes grown in the presence of [³H]leucine synthesize the new isozyme which is isotopically labeled, and pulse-chase experiments show that the two isozymes are not interconvertible. Although the two isozymes exhibit essentially identical catalytic properties, they differ markedly with regard to their stability, with the more acidic isozyme being much more labile. The lability of the more acidic isozyme may account for its low levels in most other tissues.     

Differential expression of alpha, mu and pi classes of isozymes of glutathione S-transferase in bovine lens, cornea, and retina

Isozyme characterization of glutathione S-transferase (GST) isolated from bovine ocular tissue was undertaken. Two isozymes of lens, GST 7.4 and GST 5.6, were isolated and found to be homodimers of a Mr 23,500 subunit. Amino acid sequence analysis of a 20-residue region of the amino terminus was identical for both isozymes and was identical to GST psi and GST mu of human liver. Antibodies raised against GST psi cross-reacted with both lens isozymes. Although lens GST 5.6 and GST 7.4 demonstrated chemical and immunological relatedness, they were distinctly different as evidenced by their pI and comparative peptide fingerprint. A corneal isozyme, GST 7.2, was also isolated and established to be a homodimer of Mr 24,500 subunits. Sequence analysis of the amino-terminal region indicated it to be about 67% identical with the GST pi isozyme of human placenta. Antibodies raised against GST pi cross-reacted with cornea GST 7.2. Another corneal isozyme, GST 8.7, was found to be homodimer of Mr 27,000 subunits. Sequence analysis revealed it to have a blocked amino-terminus. GST 8.7 immunologically cross-reacted with the antibodies raised against cationic isozymes of human liver indicating it to be of the alpha class. Two isozymes of retina, GST 6.8 and GST 6.3, were isolated and identified to be heterodimers of subunits of Mr 23,500 and 24,500. Amino-terminal sequence analysis gave identical results for both retina GST 6.8 and GST 6.3. The sequence analysis of the Mr 23,500 subunit was identical to that obtained for lens GSTs. Similarly, sequence analysis of the Mr 24,500 subunit was identical to that obtained for the cornea GST 7.2 isozyme. Both the retina isozymes cross-reacted with antibodies raised against human GST psi as well as GST pi. The results of these studies indicated that all three major classes of GST isozymes were expressed in bovine eye but the GST genes were differentially expressed in lens, cornea, and retina. In lens only the mu class of GST was expressed, whereas cornea expressed alpha and pi classes and retina expressed mu and pi classes of GST isozymes.     

Isolation and characterization of tissue-specific isozymes of glucosephosphate isomerase from catfish and conger.

In teleosts glucosephosphate isomerase exists as two tissue-specific isozymes. Most tissues contain the more acidic liver-type isozyme, while white muscle contains the more basic isozyme; and a few tissues contain both the liver- and muscle-type isozymes as well as a hybird. The isozymes were isolated from catfish liver and muscle and from conger muscle and shown to be homogeneous by polyacrylamide gel electrophoresis, isoelectric focusing, analytical ultracentrifugation, and rechromatography. Both isozymes are of molecular weight 132,000 (S020,w = 7.0 S) and composed of two subunits of Mr approximately 65,000. The muscle and liver isozymes were shown to have distinct isoelectric points (catfish liver = 6.2; muscle = 7.0) and amino acid compositions. Tryptic peptide maps, after S-carboxymethylation and carbamylation, revealed several distinct differences in the primary structures of the isozymes. Although the isozymes could also be distinguished on the basis of their stabilities, most of their basic catalytic properties were found to be similar. A conger was obtained which was heterozygous for the variant allele at the muscle-glucosephosphate isomerase locus. A comparison of the variant conger muscle isozyme with the wild type revealed a single altered peptide, suggesting a point mutation. The structure-function studies, as well as the genetic studies, clearly establish that the two types of isozymes are of independent genetic origin.     

Purification and characterization of two isozymes of 3-phosphoglycerate kinase from the mouse.

Two isozymes of 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), designated PGK-A and PGK-B, were purified from separate extracts of muscle and testicular tissue of DBA/2J mice, respectively. A similar procedure was used to purify the corresponding isozymes from C57BL/6J mice in order to make inter-strain comparisons. The purification involved the use of affinity chromatography with an 8-(6-aminohexyl)amino-ATP-Sepharose column and DEAE-Sephadex chromatography. Lactate dehydrogenase isozyme LDH-X was also co-purified from extract of mouse testes by this two-step procedure. The same isozyme isolated from either mouse strain was found to be identical in physical and biochemical properties. Both isozymes are monomeric as determined by gel filtration chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Furthermore, the isozymes have similar molecular weights, of 47 000 +/- 2000 and exhibit similar Km values for both coenzymes and substrate, as well as temperature dependence of enzyme activity. However, it was observed that the B isozyme is more labile than the A isozyme by denaturation at high temperature, urea and acidic pH.     

Partial purification and characterization of L-lactate dehydrogenase isozymes from sweet potato roots.

Lactate dehydrogenase [L-lactate: NAD oxidoreductase, EC 1.1.1.27] was isolated from sweet potato root tissues. Two species of the enzyme (isozymes I and II) were separated by DE-52 cellulose column chromatography from healthy, cut, and black-rot diseased tissues. Isozymes I and II were purified from healthy and diseased tissues, respectively. Reduction of pyruvate by NADH with either isozyme I or II was inhibited by pyruvate at high concentrations, by NAD+ and by several mononucleotides. Isozyme I was inhibited by a lower concentration of adenine nucleotide than isozyme II, and Km for pyruvate was increased markedly at acidic pH in the case of isozyme I, but only slightly in the case of isozyme II. The molecular weights of both isozymes were determined to be 150,000 and they were found to be charge isomers by polyacrylamide gel electrophoresis. The enzyme activity increased in response to infection by black-rot fungus but decreased in response to cutting.     

Isoelectric focusing studies of aldehyde dehydrogenases from mouse tissues: variant phenotypes of liver, stomach and testis isozymes

Isoelectric focusing techniques (IEF) were used to examine the tissue distribution and genetic variability of aldehyde dehydrogenases (AHDs) from inbred strains of mice. Twelve zones of AHD activity were resolved which were differentially distributed between tissues. Liver extracts exhibited highest activity for most enzymes, with the exception of isozymes found in stomach (AHD-4) and testis (AHD-4 and AHD-6). Genetic variants for AHD-1 (liver mitochondrial isozyme) and AHD-4 (stomach isozyme) were examined from inbred strains and F1 hybrid animals. The results were consistent with dimeric subunit structures (designated as A2 and D2 isozymes respectively). IEF patterns for activity variants of testis-specific AHD-6 were identical, with 3-banded phenotypes being observed. pI values for the AHD forms as well as for aldehyde oxidase and xanthine oxidase isozymes, which stain in the absence of coenzyme, were reported.     

Four cytosolic-type CuZn-superoxide dismutases in germinating seeds of Pinus sylvestris

A differential analysis of CuZn-superoxide dismutase (SOD. EC 1.15.1.1) isozymes after native-polyacry lamide gel elecrrophoresis (PAGE) and isoelectric focusing (IEF) indicated that germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition (3 DAI) contain five CuZn-SOD isozymes. Two isozymes co-migrated on native–PAGE but were separated after IEF. CuZn-SODs of Scots pine were purified from germinating seeds (3 DAI) by anion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The final separation of CuZn-SOD isozymes was accomplished by native-PAGE. CuZn-SOD isozymes were electroblotted and their NH₂-terminal amino acid sequence was determined. Comparisons of the amino acid sequences with sequences of CuZn-SOD isozymes from other plant sources indicated that one CuZn-SOD isozyme was of the chloroplastic type whereas the other four isozymes belonged to the cytosolic-type CuZn-SODs, The NH₂-terminal amino acid sequence of the chloroplastic CuZn-SOD and of one cytosolic-type CuZn-SOD were identical to those of two previously isolated, sequenced and localized CuZn-SOD isozymes from Scots pine needles. Two cytosolic-type CuZn-SOD isozymes showed a homology at 20 out of 21 NH₂-terminal amino acids. Mitochondria and glyoxysomes were isolated by differential and Percoll density-gradient centrifugation from germinating seeds (3 DAI). The cell fractionation experiments did not suggest that a major part of the CuZn-SOD activity in germinating seeds was derived from glyoxysomes or mitochondria.     

Analysis of Pectic Enzyme Zymograms of Fusarium Species.

Zymograms of the extracellular polygalacturonase (PG), produced by isolates of F. culmorum and F. graminearum originating from different geographic locations and different sources, were compared. PG patterns were prepared by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) of untreated fluid from liquid pectin salts cultures. There was no intraspecific variability between isolates of both Fusarium species. Electrophoretic and isofocusing PG patterns were species specific. On the basis of IEF patterns, F. culmorum (4 isozymes, estimated pI's 6.4, 6.6, 6.9, 7.1) and F. graminearum (5 isozymes, estimated pI's 6.4, 6.6, 6.9, 7.1, 7.5) could be separated from one another by the pH 7.5 PG isozyme.     

Isoelectric focusing studies of aldehyde dehydrogenases, alcohol dehydrogenases and oxidases from mammalian anterior eye tissues

1. Isoelectric focusing (IEF) and zymogram methods were used to examine the tissue distribution, multiplicity and substrate specificities of alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) and ocular oxidases (EOXs) from mammalian anterior eye tissues. 2. Baboon, cattle, pig and sheep corneal extracts exhibited high ALDH activities; the corneal ALDHs were distinct from the major liver ALDHs and distinguished by their preference for medium-chain aldehydes. 3. Baboon and pig corneal extracts also showed high ADH activities, by comparison with ovine and bovine samples. Moreover, the ADHs were distinct from the major liver isozymes in pI value and substrate specificity. 4. Mammalian lens extracts exhibited significant ALDH activity of a form corresponding to the major liver cytosolic isozyme. Minor activity of the corneal enzyme was also observed in some species. 5. Lens ADH phenotypes were species-specific, and consisted of either Class II activity (baboon and sheep), Class III ADH activity (pig), or activities of both ADH classes (cattle). 6. Lens extracts also exhibited a complex pattern of ocular oxidase (EOX) activities following IEF. 7. A role in peroxidatic aldehyde detoxification is proposed for these enzymes in anterior eye tissues.     

华南兔组织乳酸脱氢酶同工酶的研究

本文以聚丙烯酰胺凝胶电泳法分析华南兔(Lepus sinensis sinensis)11种组织乳酸脱氢酶同工酶的分布待征。分析结果:骨骼肌和肝组织5条同工酶带俱全;脑、肺、卵巢和盲肠等组织各含有4条谱带(LDH-1,-2,-3和-4);胃和肾组织含有3条谱带(LDH-1,-2,和-3);眼晶状体和睾丸组织也含有3条谱带,但前者是LDH-3,-4和-5,后者是LDH-1,-2和-5;谱带最少的是心肌组织,只有2条(LDH-1和-2)。此外,还对各组织中的亚基活性分布及电泳图谱特征进行了分析。     

Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Three alpha-naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced three major peaks of alpha-naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with pI values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25-55 degrees C). The three purified isozymes were inhibited at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DEF (10(-6) M), and PMSF (10(-6) M) indicating that they were "B" type serine esterases. Conversely, inhibition was not observed at 10(-4) M eserine, PHMB, or CaCl(2), further supporting the conclusion that the microsomal esterases were of the "B" type. None of the isozymes was inhibited by 10(-4) M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.     

COMPARATIVE ELECTROPHORESIS AND ISOZYME ANALYSIS OF SEED PROTEINS FROM CULTIVATED RACES OF SORGHUM

Comparative disc electrophoresis of acidic proteins, basic proteins, and isozymes of esterase, MDH, and peroxidase were performed with aqueous extracts of seeds from seven cultivars belonging to five races of Sorghum bicolor ssp. bicolor: bicolor, caudatum, durra, guinea, and kafir. Two disc electrophoretic systems were employed. Acidic proteins were electrophoresed in an anionic system (tris-glycine buffer, pH 8.3). Basic proteins were electrophoresed in a cationic system (β-alanin-acetate buffer, pH 4.5). Soluble proteins were stained with Coomassie brilliant blue. Isozyme activity was detected by using specific enzyme stains and substrates. Each cultivar yielded reproducible, characteristic patterns of distinct acidic and basic proteins. Cultivars belonging to the same race produced identical protein and isozyme patterns. The degree of electrophoretic similarity among races was estimated by calculating similarity index values for each of the 10 possible pairs of races. Bicolor, caudatum, durra, and guinea produced very similar acidic and basic protein patterns and esterase, MDH, and peroxidase isozyme patterns. Differences, however, were observed among all races. All of kafir patterns were significantly different from the patterns of other races. Comparative electrophoresis may provide a new source of taxonomic characters for investigating phenetic and phylogenetic relationships in Sorghum.     

普通伏翼不同组织3种同工酶的比较

采用聚丙烯酰胺不连续凝胶垂直板活性电泳,分析了普通伏翼的心、肝、肾、胸肌、肺、胃、小肠、舌8种组织的酯酶(EST)、乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)3种同工酶。结果表明:普通伏翼8种组织的3种同工酶存在差异,其分布具有明显的组织特异性,与器官或组织所执行的功能有关。     

Chinook salmon NADP+-dependent cytosolic isocitrate dehydrogenase: Electrophoretic and genetic dissection of a complex isozyme system and geographic patterns of variation

Species in the genus Oncorhynchus express complicated isocitrate dehydrogenase (IDHP) isozyme patterns in many tissues. Subcellular localization experiments show that the electrophoretically distinct isozymes of low anodal mobility expressed predominantly in skeletal and heart muscle are mitochondrial forms (mIDHP), while the more anodal, complex isolocus isozyme system predominant in liver and eye is cytosolic (sIDHP). The two loci encoding sIDHP isozymes are considered isoloci because the most common allele at one of these loci cannot be separated electrophoretically from the most common allele of the other. Over 12 electrophoretically detectable alleles are segregating at the two sIDHP* loci in chinook salmon. Careful electrophoretic comparisons of the sIDHP isozyme patterns of muscle, eye, and liver extracts of heterozygotes reveal marked differences between the tissues with regard to both relative isozyme staining and the expression of several common alleles. Presumed single-dose heterozygotes at the sIDHP isolocus isozyme system exhibit approximate 9:6:1 ratios of staining intensity in liver and eye, while they exhibit approximate 1:2:1 ratios in skeletal muscle. The former proportions are consistent with the equal expression of two loci (isolocus expression), while the latter are consistent with the expression of a single locus. Screening of over 10,000 fish from spawning populations and mixed-stock fishery samples revealed that certain variant alleles (*127, *50) are detectable only in liver and eye, while other alleles (*129, *94, and *74) are strongly expressed in muscle, eye, and liver. The simplest explanation for these observations is that the "isolocus" sIDHP system of chinook salmon (and that of steelhead and rainbow trout) results from the expression of two distinct loci (sIDHP-1* and sIDHP-2*) that have the same common allele (as defined by electrophoretic mobility). IDHP expression in skeletal muscle is due to the nearly exclusive expression of the sIDHP-1* locus, while IDHP expression in eye and liver tissues is due to high levels of expression of both sIDHP-1* and sIDHP-2*--giving rise to the isolocus situation in these latter tissues. Direct inheritance studies confirm this model of two genetically independent (disomic) loci encoding sIDHP in chinook salmon. Extensive geographic surveys of chinook salmon populations from California to British Columbia reveal marked differences in allele frequencies at both sIDHP-1* and sIDHP-2* and considerably more interpopulation differentiation than was recognized previously when sIDHP was treated as an isolocus system with only five recognized alleles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of AMP-deaminase isozymes in rat tissues

1. The distribution of AMP deaminase isozymes in rat tissues was analyzed by electrophoresis on cellulose acetate membrane, by chromatography on phosphocellulose column, and by the application of immunological technique employing specific antisera against three parental AMP deaminases (isozymes A, B and C). Skeletal muscle extracts and diaphragm extracts contain a single identical isozyme, isozyme A. The major isozyme species of liver, kidney and testes are also identical and they are isozyme B. Heart extracts contains isozyme C exclusively. Extracts of brain, lung and spleen contain five isozymes, presumably a complete set of five B-C hybrids. 2. Developmental patterns of AMP deaminase isozyme were studied. In early postnatal life, extracts of heart, liver, kidney and lung contain five isozymes similar to those observed in adult brain. During postnatal development, a shift to isozyme C occurs in heart, whereas a shift to isozyme B occurs in liver and kidney. Five isozymes in lung remain throughout development. In brain a shift of B to five isozymes is observed during development. Isozyme A is the predominant form in muscle throughout postnatal development. 3. AMP deaminase in the regenerating liver was analyzed, but the data indicated that there was no change of isozyme distribution during hepatic regeneration.     

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.