Bioluminescent Assay of Total and Brain-Specific Creatine Kinase Activity in Cerebrospinal Fluid

Abstract: A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3–0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase (CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.     

The occurrence of chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase in a range of plant species

The chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase (PGK; EC 2.7.2.3) of leaves from 18 of a broad range of 21 vascular plant species were separated by either standard or modified anion-exchange Chromatographic procedures. Immunoprecipitation of the isoenzymes with antisera raised against barley chloroplastic and cytosolic PGK isoenzymes showed that the chloroplastic isoenzymes resemble the chloroplastic isoenzymes of other species more closely than the cytosolic isoenzyme of the same species and vice versa for the cytosolic isoenzymes. Each of the two cyanobacterial species tested, yielded only a single PGK fraction on anion-exchange chromatography and gave no reaction with antisera raised against the barley isoenzymes. The cyanobacteria are presumed to contain only a single PGK which is not closely related to either of the barley PGK isoenzymes. In all of the investigated leaf extracts the catalytic activity of the cytosolic PGK was exceeded by that of the chloroplastic PGK with the ratio for many of the C₃ plants falling within the range 595 to 1585 (cytosolic: chloroplastic). The relative amounts of cytosolic PGK activity appeared to be greater in older leaves, in C₄ and CAM plants and in ferns.Abbreviations CAM crassulacean acid metabolism - pgk phosphoglycerate kinase This work was supported by the Science and Engineering Research Council (grant no. GR/E54504) and also the King's College London Research Strategy Fund.     

白细胞酯酶同工酶对胃癌诊断价值的探讨

本文应用薄平板等电聚焦电泳法对15例未经手术治疗的胃癌患者进行了外周血白细胞酯酶同工酶的分离.结果:正常人外周血白细胞酯酶同工酶显示12条带,等电点(PI)为3.73～7.80.胃癌患者显示11条带,与正常相比第十二条带缺如,PI为3.80～7.90.同工酶各分带与正常相比,酶活性发生明显异常(P<0.01).提示;胃癌患者酯酶同工酶的改变可能是胃癌早期诊断的酶学指标.     

Segregation and linkage relationships of isoenzymes in shortleaf pine (Pinus echinata Mill.)

 Segregation and linkage relationships were analyzed between 28 isoenzyme loci in ten natural stands representing much of the natural range of Pinus echinata Mill. (shortleaf pine). A total of 203 possible two-locus combinations were tested. Three linkage groups were revealed in this study at a linkLOD of 4.0. The first linkage group (A) consisted of Pgi and Adh-1; Gdh, Idh, Skdh-2, G6pd-2 and Aco were mapped to the second linkage group (B); the third group (C) had 2 loci: Mdh-2 and Mdh-3. A moderate linkage between Mnr-2 and Dia-2 and weak linkages between Mnr-1 and Dia-1, and Got-2 and 6pgd-2 were also detected. The significance of these results in shortleaf pine is discussed and compared to linkage maps previously reported in other conifers, including pines. Received: 7 April 1997 / Accepted: 25 April 1997     

Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human beta 1 beta 1 and horse EE alcohol dehydrogenases.

The horse EE and human β₁β₁ alcohol dehydrogenase isoenzymes have almost identical protein backbone folding patterns and contain 2 tryptophans per subunit (Trp-15 and Trp-314). Tyr-286, which had been proposed to quench the fluorescence of Trp-314 by resonance energy transfer at alkaline pH in EE, is substituted by Cys in β₁β₁. The proposed role of Tyr-286 in pH-dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of β₁β₁ is not substantially quenched at alkaline pH. Tyr-286 had also been implicated in the quenching of Trp-314 upon formation of the EE-NAD⁺-trifluoroethanol ternary complex. However, β₁β₁ exhibits the same extent of tryptophan fluorescence quenching as EE upon complexation, which strongly suggests that Tyr-286 is not involved in ternary complex quenching.     

Fertile progeny of a hybridization between soybean [Glycine max (L.) Merr.] and G. tomentella Hayata

Summary A colchicine-doubled F₁ hybrid (2n=118) of a cross between PI 360841 (Glycine max) (2n=40) x PI 378708 (G. tomentella) (2n=78), propagated by shoot cuttings since January 1984, produced approximately 100 F₂ seed during October 1988. One-fourth of the F₂ plants or their F₃ progeny have been analyzed for chromosome number, pollen viability, pubescence tip morphology, seed coat color, and isoenzyme variation. Without exception, all plants evaluated possessed the chromosome number of the G. max parent (2n=40). Most F₂ plants demonstrated a high level of fertility, although 2 of 24 plants had low pollen viability and had large numbers of fleshy pods. One F₂ plant possessed sharp pubescence tip morphology, whereas all others were blunt-tipped. All evaluated F₂ and F₃ plants expressed the malate dehydrogenase and diaphorase isoenzyme patterns of the G. max parent and the endopeptidase isoenzyme pattern of the G. tomentella parent. Mobility variants were observed among progeny for the isoenzymes phosphoglucomutase, aconitase, and phosphoglucoisomerase. This study suggests that the G. Tomentella chromosome complement has been eliminated after genetic exchange and/or modification has taken place between the genomes.Journal Paper No. J-13776 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, USA, Project 2763     

The development of the activity of the chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase during barley leaf ontogenesis

The catalytic activities of the chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase (PGK; EC 2.7.2.3) have been followed during the development of the first leaf of barley (Hordeum vulgare L.) grown for 7 d in darkness followed by transfer to continuous illumination. The investigation has included both the study of a standard leaf section, measured from the leaf tip, over the whole life of the leaf and the study of serial sections of leaf, measured from the leaf base, at a standard sampling time. The results of both approaches were fully compatible. As the catalytic activity of each isoenzyme in the standard assay is directly proportional to the amount of isoenzyme protein present, the catalytic activities may be interpreted wholly in terms of enzyme synthesis and degradation. Both isoenzymes are synthesized in darkness and in etiolated barley are present at a ratio of about 2674 for the cytosolic to chloroplastic isoenzymes. Illumination results in a fivefold or greater increase in chloroplast PGK over a number of days with little change of the cytosolic isoenzyme, resulting in an eventual ratio of cytosolic to chloroplastic isoenzymes approaching the green-leaf value of about 991. Prior to any detectable onset of senescence a 15-fold increase in cytosolic isoenzyme commenced while the amount of chloroplast PGK remained constant. It is suggested that the increased cytosolic PGK may be involved in the export of carbohydrate reserves (starch) prior to leaf senescence. Both isoenzymes subsequently decline in parallel to total protein and chlorophyll in the course of senescence.Abbreviations DHAP reductase dihydroxyacetone-phosphate reductase - GS glutamine synthetase - LHCP light-harvesting chlorophyll-a/b-binding protein - PGK phosphoglycerate kinase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by the Science and Engineering Research Council (grant no. GR/E54504).     

Alterations of Na,K-ATPase Isoenzymes in the Rat Diabetic Neuropathy: Protective Effect of Dietary Supplementation with n-3 Fatty Acids

Abstract: Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase α subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of α1- and α3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.     

Ribonuclease S-peptide as a carrier in fusion proteins.

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).     

Antioxidant and detoxifying fish enzymes as biomarkers of river pollution

The activity of several antioxidant and detoxifying enzymes, superoxide dismutase SOD , GSH peroxidase GSHPx , GSSG reductase GSR and GSH S transferase GST , the contents of thiobarbituric acid reactive substances TBARS , and the SOD and GST isoenzyme patterns were studied in the livers of chubs Leuciscus cephalus from reference river areas and polluted urban sites. Livers of polluted fish contained higher concentrations of transition metals, especially copper and iron. Total GSHPx activity was 1.8 fold higher in the polluted fish than in reference animals, while the SOD and GSR activities and the TBARS content were not significantly changed. Three new SOD isoforms pI 4.45, 5.1, 5.2 and a higher intensity of the band pI 4.2 were observed after isoelectrofocusing of polluted fish extracts. Total GST activity was higher in fish from polluted areas. The GST isoenzyme pattern was studied using subunit specific substrates DCNB, EPNP, EA, NPB, NBC and by Western blot using antibodies specific to rat GST subunits 1, 8 Alpha class , 3 Mu class and 7 Pi class . Reference and polluted fish lacked cross reactivity towards Alpha class GSTs. Reference fish displayed weaker cross reactivity towards CST 7 and 2.3 fold lower activity with EA, while higher cross reaction with GST 3 was observed in polluted fish.