Genotype dependent radiosensitivity of autotetraploids in Trigonella foenum-graecum L.

Summary Primary trisomics were used to locate the structural loci coding for particular forms of the dimeric enzymes phosphoglucoisomerase and glutamate oxaloacetate transaminase in Lolium perenne. The polymorphy of these loci enabled triallelic trisomics to be produced. Each locus could thus be directly assigned to a particular chromosome without the need to examine segregant progeny. The loci for GOT/3 and PGI/2 were found to be located on chromosomes 2 and 6 respectively.     

Effects of Selection for Dark Respiration Rate on Enzyme Genotypes in Lolium perenne

Two lines of perennial ryegrass (Lolium perenne) that had previouslybeen selected for ‘fast’ and ‘slow’rates of leaf dark respiration were examined to determine whetherselection had differentiated allelic or genotypic frequenciesof five polymorphic enzyme loci, and determine whether respirationrates differed among genotypes. The lines were differentiatedfor genes coding for phosphoglucose isomerase (PGI) and UDP-glucosepyrophosphorylase (UDP). Respiration rates were heterogeneousamong UDP and 6-phosphogluconate dehydrogenase (6PGD) genotypes.Founder effects may have produced the genetic differentiationof lines at the PGI locus as we detected no differences amonggenotypes for respiration rates. The genetic differentiationamong lines for UDP was consistent with differences in darkrespiration among genotypes. Although we detected large differences(75%) in dark respiration rate among 6PGD genotypes in the fastrespiring line, selection did not differentiate the lines atthis locus. Lolium perenne L., perennial ryegrass, enzyme polymorphisms, dark respiration rate     

Development of phosphogluco-isomerase isozymes in perennial ryegrass (Lolium perenne)

The development of isozymes of phosphogluco-isomerase (PGI; D-glucose-6-phosphate ketol isomerase EC 5.3.1.9.) in perennial ryegrasses was followed from dry seed through to leaf senescence using starch gel electrophoretic separations. Root isozymes were also examined. Two forms of the enzyme were found, one (PGI/2) being present in all tissues and at all stages of the life cycle. The other (PGI/1) had two zones of activity, one of which was detected only in light-exposed tissue. Normal development of this form could be inhibited by growing seedlings on distilled water. Some alleles of PGI/2 not previously reported for ryegrasses are also described.     

Phosphoglucose isomerase from bananas: partial characterization and relation to main changes in carbohydrate composition during ripening.

Some characteristics of phosphoglucose isomerase (PGI, EC 5.3.1.9) from banana were measured during fruit ripening of three banana cultivars. In banana, PGI was present as two dimeric isoenzymes, named PGI1 and PGI2, which had similar native molecular masses but differed in relation to heat stability and isoelectric point. Total PGI activity showed a distinct two-step change during fruit ripening. Before the climacteric period, PGI activity gradually decreased with the starch content, then its activity began to increase with sucrose accumulation. The ratio of PGI1, and PGI2 was constant, indicating that both enzymes would be involved in starch degradation and sucrose synthesis. PGI activity and changes in carbohydrate composition suggests the existence of some control to fit the requirements of the intense carbon flow from starch to sucrose.     

Identification and mode of action of self-compatibility loci in Lolium perenne L

The two-locus gametophytic incompatibility system in perennial ryegrass (Lolium perenne L.) is not always fully effective: obligate selfing of plants sieves self-compatible pollen mutants, and self-fertility becomes fixed in subsequent generations. Self-compatibility (SC) was investigated in an F2 family. In vitro self-pollinations were analysed and recorded and plants were classified as being either partially or fully compatible. Distorted segregation ratios of markers on linkage group (LG) 5 were found, which indicate the possible presence of a gametophytic SC locus. Interval linkage analysis of pollen compatibility after selfing confirmed that this distortion was due to a locus (T) analogous to the S5 locus of rye. However, even though markers in this region were, on average, less than 1 cM apart, the minimum number of plants possessing the unfavoured allele was never less than 6% for any marker locus. We proved that this was because of the presence of another SC locus, exhibiting gametophytic selection, segregating in this population and identified by interval mapping analysis of compatibility classes of in vitro self-pollinations. This locus was located on LG1, and probably corresponds to the S locus. We show that the T locus, a relic of a multilocus system, functions through interaction with the S locus: F2 segregation of incompatibility phenotypes and linked markers demonstrated that the S/t pollen genotype combination, expected to be compatible on selfing, was sometimes incompatible. Further evidence is presented to show that this interaction must be dependent on yet another locus located on LG2. A prime candidate would be the Z incompatibility locus.     

Isolation and molecular analysis of the phosphoglucose isomerase structural gene of Saccharomyces cerevisiae

Summary The PGI1 gene of Saccharomyces cerevisiae coding for the glycolytic enzyme phosphoglucose isomerase has been cloned by complementation of a mutant strain (pgi1) with a strongly reduced phosphoglucose isomerase activity. A genomic library constructed in the yeast multicopy vector YEp13 (Nasmyth and Tatchell 1980) was used. Four plasmids containing an overlapping region of 4.1 kb were isolated and characterized by restriction endonuclease mapping. Southern analysis of genomic digests prepared with different restriction enzymes confirmed the same pattern for the chromosomal sequences. Transformants with the isolated plasmids had a phosphoglucose isomerase activity increased by a factor of 7. The cloned sequence hybridized to a constitutively synthesized 2.2 kb RNA in Northern analysis. The coding region includes a 2.05 kb EcoRI fragment common to all four inserts. A fragment including part of the PGI1 region was subcloned into vector YRp7 and used to induce integration at the PGI1 locus. Genetical and Southern analysis of stable transformants showed that single as well as tandem integration took place at this locus. This showed that the PGI1 gene had been isolated. Finally, and in contrast to the results of Kempe et al. (1974a, b) who reported three isoenzymes in yeasts, only one copy of the PGI1 gene per genome was found in several laboratory strains tested by Southern analysis.     

The renal haemodynamic and excretory actions of prostacyclin and 6-oxo-PGF1 alpha in anaesthetized dogs.

Intrarenal arterial (i.a.) infusions of prostacyclin (PGI2) at 30-300 ng/min to anaesthetized dogs reduced renal vascular resistance (RVR) and filtration fraction (FF), increased mean renal blood flow (MRBF) but did not alter mean arterial pressure (MAP)or glomerular filtration rate (GFR). The urinary excretion of sodium (UNaV), potassium (UKV) and chloride ions (UC1V) were increased through inhibition of net tubular ion reabsorption. PGI2 (3000 ng/min, i.a.) reduced MAP and increased heart rate. Intravenous (i.v.) infusions of PGI2 (3000 gn/min) reduced MAP, GFR, FF, urine volume and ion excretion, with elevation of heart rate. The measured variables were unaltered by 6-oxo-PGF1 alpha (10,000 ng/min i.a.). Treatment of the dogs with the PG synthetase inhibitor meclofenamic acid (2.5 mg/kg i.v.) did not antagonise the elevation of MRBF to PGI2 (300 ng/min i.a.). Thus the renal effects of PGI2 were due to a direct action rather than through conversion to 6-oxo-PGF1 alpha or through stimulation of endogenous renal PG biosynthesis and release.     

Competitive inhibition of spinach leaf phosphoglucose isomerase isoenzymes by erythrose 4-phosphate

In plant cells, the reversible isomerization between fructose 6-phosphate (Fru6P) and glucose 6-phosphate (Glc6P) is catalyzed by a cytosolic and a chloroplastic isoenzyme of phosphoglucose isomerase (PGI, EC 5.3.1.9). The extractable activities of both PGI isoenzymes are in large excess compared with the flux required for product synthesis, but the measured Glu6P/Fru6P ratio in illuminated chloroplasts and in whole leaves is always displaced from equilibrium. Cytosolic (PGI 2) and stromal (PGI 1) isoenzymes were purified from spinach leaves and used to investigate the possibility of metabolic regulation at this step. Several metabolites were found to inhibit PGI, but within the physiological concentration range, only erythrose 4-phosphate (Ery4P) inhibited significantly. The inhibition was competitive, with K_i values below 10 μM for PGI 2 and 1. The physiological significance of the inhibition of PGI by Ery4P was assessed in isolated intact spinach chloroplasts. We conclude that, in vivo, this inhibition is probably responsible for the observed displacement from equilibrium in the chloroplasts, but limits the carbon flow towards starch synthesis only when Fru6P is low. In contrast, the inhibition by Ery4P is unlikely to play any role in the cytosolic carbon metabolism because both Fru6P concentration and PGI activity, are much higher than in the chloroplast stroma.     

Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoglucose Isomerase from Bananas: Partial Characterization and Relation to Main Changes in Carbohydrate Composition during Ripening

Some characteristics of phosphoglucose isomerase (PGI, EC 5.3.1.9) from banana were measured during fruit ripening of three banana cultivars. In banana, PGI was present as two dimeric isoenzymes, named PGI₁ and PGI₂, which had similar native molecular masses but differed in relation to heat stability and isoelectric point. Total PGI activity showed a distinct two-step change during fruit ripening. Before the climacteric period, PGI activity gradually decreased with the starch content, then its activity began to increase with sucrose accumulation. The ratio of PGI₁, and PGI₂ was constant, indicating that both enzymes would be involved in starch degradation and sucrose synthesis. PGI activity and changes in carbohydrate composition suggests the existence of some control to fit the requirements of the intense carbon flow from starch to sucrose.     

Influence of verapamil on central and peripheral effects of prostacyclin on circulatory system in rats

The influence of verapamil on cardiovascular effects of prostacyclin (PGI2) in rats was examined. PGI2 administered into the lateral brain ventricle (i.c.v.) or intravenously (i.v.) in a dose of 2.7 x 10(-8)mol evoked hypotension and tachycardia. Pretreatment with verapamil in a dose of 2.0 x 10(-5)mol/kg given intraperitoneally (i.p.) diminished hypotensive effect of PGI2 i.c.v. as well as inhibiting the influence of PGI2 i.c.v. and i.v. upon the heart rate. Bolus injection of PGI2 in a dose of 2.7 x 10(-10), 2.7 x 10(-9) or 2.7 x 10(-8)mol evoked biphasic inotropic and chronotropic effects on isolated rat heart. Short-term increase of the contractile force together with bradycardia and afterwards long-lasting decrease of contractility with sustained, slight tachycardia were observed. Verapamil in a concentration of 1.0 x 10(-6)M blocked biphasic inotropic effect and bradycardia after PGI2 administration. Because some central and peripheral cardiovascular effects of PGI2 were inhibited by verapamil, it is concluded that PGI2 may participate in transmembrane calcium ions movements.     

Comparison of gastric and intestinal antisecretory and protective effects of prostacyclin and its stable thia-imino-analogue (Hoe 892) in conscious rats

The effects of prostacyclin (PGI2) and its stable thia-thimo-analogue (Hoe 892) on gastric and intestinal secretions and gastric mucosal lesions have been determined in conscious rats. Both PGI2 and Hoe 892 given subcutaneously (s.c.) reduced dose-dependent gastric acid secretion, the ID50 (dose producing 50% inhibition) being about 48.6 and 11.8 micrograms/kg, respectively. In contrast, intragastric (i.g.) PGI2 and Hoe 892 did not cause any change in gastric acid secretion at doses ranging from 1 to 100 micrograms/kg. Both PGI2 and Hoe 892 reduced significantly intestinal fluid secretion (antienteropooling activity). PGI2 and Hoe 892 given i.g. or s.c. reduced dose-dependent gastric ulcer formation induced by acidified aspirin (ASA), Hoe 892 being somewhat less potent than PGI2. Both PGI2 and Hoe 892 were equally effective against gastric mucosal necrosis induced by absolute ethanol and this effect was observed both after i.g. and s.c. administration of these agents. We conclude that stable thia-imino-PGI2 analogue, Hoe 892, has similar gastric and intestinal antisecretory and protective activity as PGI2 and may be useful in the prevention of gastric damage by various noxious agents.     

Isoenzymes of phosphoglucomutase from human red blood cells: isolation and kinetic properties

A procedure has been developed for the purification of phosphoglucomutase from human red cell (phenotype PGM1 a1 or a3) lysates. It yields homogeneous isoenzyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci with distinctive pI (from 6.07 to 5.29). There are substantial differences between PGM1 and PGM2 isoenzymes, having single polypeptide chains of 58,500 and 69,000 Mr respectively and showing different thermostability. The kinetic properties of all the isoenzymes for the phosphoglucomutase reaction are essentially the same (apart from the specific activity of 1089-1263 units/mg for PGM1 forms vs 37-42 units/mg for PGM2 forms), but there are striking differences in substrate specificity. In fact the products of PGM1 locus are "true" phosphoglucomutases, being specific to mutate glucose monophosphates, whereas the PGM2 forms also display phosphoribomutase and glucose 1,6-bisphosphate synthetic activities. Some kinetic properties of these "side activities" are also reported.     

Separation and partial characterization of multiple forms of rat liver alcohol dehydrogenase

Rat liver alcohol dehydrogenase was purified and four isoenzyme forms, demonstrated by starch gel electrophoresis, were separated by O-(carboxymethyl)-cellulose chromatography. Each of the isoenzymes had a distinct isoelectric point. All isoenzymes were active with both ethanol (or acetaldehyde) and steroid substrates, and had similar Michaelis-Menten constants for each of the substrates and coenzymes studied. The three isoenzymes with the lowest migration toward the cathode exhibited the same pH optimum of 10.7 for ethanol oxidation, a greater activity with 5 beta-androstan-3 beta-ol-17-one than with ethanol as a substrate, and an unchanged electrophoretic mobility following storage in the presence of 100 microM dithiothreitol. By contrast the isoenzyme with the highest mobility toward the cathode exhibited a pH optimum of 9.5 for ethanol oxidation, a low steroid/ethanol ratio of activity, and converted to the migrating pattern of the two isoenzymes with intermediate mobility when stored. The similarities between the isoenzymes of rat liver alcohol dehydrogenase differ considerably from differences in substrate specificity exhibited by isoenzymes of horse liver alcohol dehydrogenase.     

Leaf Development in Lolium temulentum L.: Progressive Changes in Soluble Polypeptide Complement and Isoenzymes

Ougham, Helen J., Jones, Thomas W. A. and Evans, Mair LL. 1987.Leaf development in Lolium temulentum L.: progressive changesin soluble polypeptide complement and isoenzymes.—J. exp.Bot. 38: 1689–1696. The spectrum of soluble polypeptides extracted from segmentsof the developing 4th leaf of Lolium temulentum simplified withincreasing distance from the leaf base. Most of the metabolicallyimportant isoenzymes analysed also exhibited gradients of activitywith respect to distance from the base, and in some cases twoor more contrasting gradients were observed for a given enzyme. Key words: Gradients, isoenzymes, leaves, Lolium temulentum,, soluble polypeptides     

Carbonic anhydrase isoenzymes in the erythrocytes and uterus of the rabbit

1. Two forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from rabbit erythrocytes and two forms from rabbit uterine tissue (endometrium) in the progestational stage of pregnancy (days 6-8 of gestation). Separation of the isoenzymes was achieved by ion-exchange chromatography, preparative polyacrylamide-gel electrophoresis and isoelectric focusing. A comparison was made of the general properties and kinetic behaviour of the purified isoenzymes. 2. Although indistinguishable in terms of molecular weight and zinc content the isoenzymes were very different as catalysts of the hydration of carbon dioxide. The two erythrocyte isoenzymes, found in almost equal amounts, differed more than 100-fold in specific activity. Of the two isoenzymes prepared from either endometrial or entire uterine homogenates one was kinetically indistinguishable from the erythrocyte high-activity form, whereas the other, also possessing high activity, was found only in the endometrial or uterine tissue. Present evidence suggests that the former isoenzyme originated from residual blood contaminating the tissue homogenates, and that a marked rise in the content of the latter isoenzyme accounts for the increase in rabbit endometrial carbonic anhydrase activity that previously has been observed in early pregnancy. 3. Minor forms of the erythrocyte isoenzymes, having a characteristic quantitative and electrophoretic relationship to one another, were occasionally produced during purification. 4. The actions were investigated of the inhibitors acetazolamide (5-acetamido-3,4-diazole-1-thia-2-sulphonamide), 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxyzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydration of carbon dioxide and the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was superior to the high-activity form as a catalyst of beta-naphthyl acetate hydrolysis.     

柚品种的等位酶变异研究

研究了柚的48个品种的等位酶变异,利用等位酶分析技术对柚的酯酶（EST）,6－磷酸葡萄糖异的酶（PGI）,6－磷酸葡萄糖变位酶（PGM）,莽草酸脱氢酶（SKD）,超氧化物歧化酶（SOD）共5个酶系统的10个等位酶基因座进行了分析,除PGI－1,PGI－2两个基因座外,其它8个均为多态性基因座;10个等位酶基因座共观察到的等位基因25个,平均每个基因座的有效等位基因数目为1．55,基因多样度0．2805,柚的品种间具有较为丰富的等位酶标记遗传多样性,但柚类种质资源群体总的遗传多样性水平偏低。柚的较低的有效等位基因数目与基因多样度可能由于人工选择及资源流失造成。     

Allozyme variation within and among populations of onion (Allium cepa L.) from West Africa

Local germplasm of onion (Allium cepa L.) in West Africa is threatened by extinction. Sixteen populations of onion collected in five countries in West Africa were investigated for isozyme polymorphism using four polymorphic enzyme systems (ADH, MDH, 6-PGDH and PGI) among nine enzyme systems assayed. This is the first report on the genetic diversity of local landraces of onion. The inheritance of two dimeric enzyme systems PGI and MDH was demonstrated using F₂ progeny arrays. The PGI system revealed a single locus with three alleles, and the MDH system revealed three loci with four alleles. Four polymorphic systems revealed nine alleles (adh-a1 and a2, mdh-c1 and c2, 6-pgdh-a1 and a2, and pgi-a1, a2 and a3) in the 16 local populations observed. The mean number of alleles per polymorphic locus was 2.25, and 67% of the alleles were present in all populations. Allele 6-pgdh-a2 was present in only two landraces (from Niger and Nigeria); it is considered to be a rare allele (frequency approximately 2%). Among the 16 populations, within-population diversity was greater (90%) than between-population diversity (10%). Genetic distance analyses showed an aggregate of all populations except for two, which originated from Nigeria, an English-speaking country. Received: 24 August 1995 / Accepted: 26 February 2001     

滇产回心草及回心康抗缺血心肌脂质过氧化作用及对前列环素／血栓素的影响

采用结扎大鼠冠脉造成急性心肌缺血模型,动态观测Ⅱ导联心电图ST段的变化,以S-T段上移为指标反映缺血程度,观察了滇产回心草及回心康对急性心肌缺血损伤的保护作用,同时检测血清SOD、MDA、PGI2和TXA2.结果表明,回心草及回心康均能显著减少S-T段上移,均以2 g/kg组为显著;其1 g/kg及2 g/kg均使心肌梗塞范围缩小(p＜0.05,p＜0.01);均使心肌缺血大鼠血清SOD显著提高(p＜0.05及p＜0.01);MDA显著降低(p＜0.05,p＜0.01).回心草及回心康还能提高大鼠血清PGI2水平,降低TXA2含量,以回心草2 g/kg及回心康2 g/kg为显著(p＜0.05),呈剂量依赖性.实验结果提示,回心草及回心康均具有抗心肌缺血及抗脂质过氧化作用,其提高PGI2/TXA2可能与其抗心肌缺血作用机制有关.     

Ca2(+)-dependent synthesis of prostaglandin I2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.