The Two Myosin Isoenzymes of Chicken Anterior Latissimus dorsi Muscle Contain Different Myosin Heavy Chains Encoded by Separate mRNAs

Abstract. The two myosin isozymes (SM1 and SM2) of the anterior latissimus dorsi muscle of the chicken change in relative concentration during development. As SM1 decreases from 13 days of embryonic growth through 1 year of adult maturation, SM2 increases. In the adult muscle SM2 accounts for over 95% of the total myosin. The myosin heavy chains of the two isozymes are distinctly different and may be separated from each other by 5% SDS polyacrylamide gel electrophoresis. The faster migrating myosin heavy chain is identified as originating from SM1 and the slower migrating myosin heavy chain from SM2 myosin isozymes. The myosin heavy chains change in relative concentration during development exactly parallel with changes in SM1 and SM2 isozyme levels. Peptide map analysis also reveals that SM1 myosin heavy chains and SM2 myosin heavy chains are distinctly different. When RNA from the ALD muscle is added to reticulocyte lysate protein synthesizing systems the translation products are shown to include both SM1 and SM2 myosin heavy chains. These comigrate exactly on 5% SDS polyacrylamide gels with authentic counterparts from ALD muscle. Finally, when peptide maps of SM1 and SM2 myosin heavy chains synthesized in the reticulocyte lysate are compared they are again found to be distinctly different and each is identical to a peptide map of respective authentic SM1 and SM2 myosin heavy chains. It is concluded that the myosin heavy chains of SM1 and SM2 myosin isozymes of ALD muscle have different primary structures and that they are encoded by two distinctly different mRNAs.     

Glucosyltransferase isozymes forming storage glucan in prochloron, a prokaryotic green alga

Since the prokaryotic, green marine alga Prochloron has not, as yet, been cultured, lyophilized cells were used in a microadaptation of polyacrylamide gel electrophoresis (PAGE) in order to isolate the glucosyltransferase isozymes. The pattern obtained with these capillary gels was identical with those of cyanophytes. Besides two phosphorylase and synthase isozymes, three branching isozymes of the b.e. type were found to be present.     

Determination of the molecular weights of ribonuclease isozymes in a cell-free crude extract of Dictyostelium discoideum, by activity-staining of gels after SDS-PAGE.

1. In a previous report we described three isozymes of intracellular ribonuclease in Dictyostelium discoideum, which were found in vegetative cells. Here we report that the molecular weights of the three isozymes from vegetative cells. 2. They are 14.3 kDa, 60 kDa and 80 kDa, as determined by activity-staining of gels after SDS-PAGE. 3. For renaturation of ribonucleolytic activity from D. discoideum cells after SDS-PAGE, fibrinogen-containing gels were used and gels were washed in aqueous isopropanol to remove detergent. Results of studies by this method suggest that each of these isozymes is composed of only a single polypeptide. 4. The effect of the buffer system on this technique is discussed.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

Alteration of 6-phosphofructo-1-kinase isozyme pools during heart development and aging

The nature of 6-phosphofructo-1-kinase isozyme pools in fetal, neonatal, young adult (3 months), and aged (30 months) rat hearts was studied using chromatographic and immunological techniques. Furthermore, the changing subunit composition of each isozyme pool was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 6% slab gels and by immunoblotting with subunit-specific antibodies. Although all three subunit types were expressed in heart throughout life, total activity and the nature of the isozyme pools varied during neonatal development and in aged heart. In fetal heart, the complex tetramers containing all three subunits appeared to be the major isozyme types. As the heart matured to the young adult stage, the M-type subunit increased over 6-fold; whereas the changes in the other two subunits were considerably less. These data indicate that during neonatal heart maturation the isozymic pools progressively exhibited increased amounts of the tetrameric forms containing two or more M-type subunits. In aged heart relative to the young adult (3 months) heart, the total activity and proportion of M-type subunit in the isozymes were decreased; and consequently, the amounts of the M-rich isozymes were decreased. The shifts in the types of isozymes during heart maturation and subsequent aging were primarily due to changes in availability of the M-type subunit to participate in random assembly of the tetrameric isozymes.     

Diagnostic assays based on esterase-mediated resistance mechanisms in western corn rootworms (Coleoptera: Chrysomelidae)

Resistance to methyl-parathion among Nebraska western corn rootworm, Diabrotica virgifera virgifera LeConte, populations is associated with increased hydrolytic metabolism of an organophosphate insecticide substrate. An electrophoretic method to identify resistant individuals based on the staining intensity of esterase isozymes on nondenaturing polyacrylamide gels was developed. Three groups of esterases (I, II, and III) were visible on the gels, but only group II esterase isozymes were intensified in resistant populations. A total of 26 and 31 field populations of western corn rootworms from Nebraska (in 1998 and 1999, respectively) were assessed with nondenaturing polyacrylamide gel electrophoresis (PAGE) assays and diagnostic concentration bioassays. Significant correlations were observed between the two diagnostic assays. Group II esterase isozymes provide a reliable biochemical marker for detection of methyl-parathion resistance in individual western corn rootworms and a tool for monitoring the frequency of resistant individuals in field populations.     

罗氏沼虾个体发育早期的同工酶研究

采用聚两燃酰胺梯度凝胶电泳技术,对罗氏沼虾个体发育早期9个时期的八种同工酶系统（EST、ALP、AMY、GDH、MDH、LDH、SOD、ME)进行研究,结果表明：SOD、ME在早期发育过程中酶谱相对稳定,SOD表现为三条谱带,ME表现为两条谱带;面EST、ALP、AMY、GDH、MDH、LDH则随发育其酶谱表现出明显差异,酶谱渐趋复杂。     

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.     

Extracellular Enzyme Activities during Lignocellulose Degradation by Streptomyces spp.: A Comparative Study of Wild-Type and Genetically Manipulated Strains

The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Isoenzymes were analyzed by polyacrylamide gel electrophoresis and activity staining on the gels. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

The purification of nuclease-free T4-RNA ligase.

RNA ligase has been highly purified in good yields from bacteriophage T4-infected Escherichia coli by a rapid and reproducible procedure. The enzyme is free of phosphomonoesterase and ribonuclease activities and is therefore suitable for the synthesis of oligoribonucleotides and for the labeling of the 3'-terminus of RNA. Greater than 90% of the protein in the enzyme preparation migrates as a single band on gradient polyacrylamide gels containing sodium dodecyl sulfate during electrophoresis. For use as a DNA synthesis reagent the enzyme may be reliably freed of deoxyribonuclease activity by an additional chromatographic procedure using a commercially avialable resin.     

以靛酚乙酸酯为底物的酯酶同工酶显色方法的研究

建立一种以靛酚乙酸酯为底物的酯酶同工酶的显色新方法。酯酶样品的聚丙烯酰胺凝胶电泳（PAGE）凝胶用磷酸缓冲液漂洗约10min后,浸入含有0.002%靛酚乙酸酯的溶液显色5～10min,可显出清晰的蓝色酯酶带。先将酯酶凝胶板浸于有机磷农药溶液中,然后再用靛酚乙酸酯显色液显色,比较同工酶谱,从同工酶带由深蓝色变为浅蓝色的颜色变化,可以看出对有机磷农药敏感的同工酶所受到的抑制程度。     

Analysis of maize alcohol dehydrogenase by native-SDS two dimensional electrophoresis and autoradiography

Summary Alcohol dehydrogenase isozyme proteins were characterized by visualization on two dimensional polyacrylamide gels. Native first dimension electrophoresis separates isozymes by size and charge, while preserving enzyme activity and subunit interactions. SDS electrophoresis in the second dimension breaks subunit interactions and separates polypeptides primarily by molecular weight. Results revealed that ADH2 monomers are larger in molecular weight than ADH1 monomers. An EMS induced Adhl mutant was found to produce ADH1 monomers of reduced molecular weight. Autoradiography revealed that only a few proteins (five or six) including ADH1 and ADH2 actively incorporate labelled amino acids after prolonged anaerobiosis.     

A role for glyceraldehyde-3-phosphate dehydrogenase in the development of thermotolerance in Xenopus laevis embryos

During heat shock, Xenopus laevis embryos exhibit an increase in the rate of accumulation of lactate and a loss of ATP relative to non-heat-shocked control embryos. These results suggest that heat shock stimulates a shift in energy metabolism to anaerobic glycolysis while at the same time causing an increase in the demand for ATP. We have evidence indicating that the embryo may meet such demands placed on it by increasing the levels of some glycolytic enzymes. In this report, we show that heat shock stimulates increases in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase [( EC 1.2.1.12] GAPDH). The specific activity of GAPDH shows a significant increase after heat shock, which correlates with the accumulation of GAPDH in heat-shocked embryos as detected by immunoblotting. Increases in GAPDH-specific activity are variable, however, and are inversely proportional to the levels of specific activity in control embryos; i.e., constitutive enzyme activity. We further analyzed the heat-enhanced accumulation of GAPDH by electrophoretically separating GAPDH isozymes on nondenaturing polyacrylamide gels. Control embryos exhibit a single isozyme of GAPDH, whereas heat-shocked embryos exhibit two isozymes of GAPDH. When these isozymes are labeled with [35S]methionine, separated by nondenaturing gel electrophoresis, and analyzed by fluorography, a heat-shock protein is found to comigrate with the isozyme unique to the heat-shocked sample. Enzyme activity assays at different temperatures suggest that this isozyme has optimum enzymatic activity only at heat-shock temperatures. We have correlated a 35-kD heat-shock protein (hsp35) with GAPDH using the following evidence: this hsp comigrates with GAPDH on one-dimensional SDS polyacrylamide gels; heat-enhanced increases in GAPDH specific activity correlate with hsp35 synthesis; and hsp35 and GAPDH have similar peptide maps. This relationship also provides a compelling explanation for the restriction of hsp35 synthesis to the vegetal hemisphere cells of heat-shocked early gastrulae reported previously (Nickells, R. W., and L. W. Browder. 1985. Dev. Biol. 112:391-395).     

Purification and characterization of a lysosomal form and a variant form of beta-glucuronidase from the rat basophil leukaemia tumour.

Two isoenzyme of beta-glucuronidase from a rat basophil leukaemia tumour were co-purified 4067-fold by (NH4)2SO4 precipitation and sequential chromatography on concanavalin A--Sepharose, Sephadex G-200, DEAE-cellulose, CM-cellulose and phosphocellulose. The purity of the mixture was established by the coincidence of the peaks of enzyme activity and protein at a molecular weight of 300 000 on Bio-Gel P-300, the presence of only two protein bands, both of them enzymically active, in polyacrylamide gels after electrophoresis under non-denaturing conditions, and the presence of a single subunit species, of mol.wt. 75 000, after electrophoresis in polyacrylamide gels under a denaturing conditioning. The major isoenzyme co-migrated with the L form from rat liver during electrophoresis in alkaline polyacrylamide gels, whereas the minor isoenzyme migrated more rapidly than either the lysosomal form or the rat liver microsomal form and was designated the tumour (T) isoenzyme. A mixture of the purified isoenzymes from two preparations had an average specific activity of 1389 units/mg for phenolphthalein beta-D-glycopyranosiduronic acid. The L and T isoenzymes, which had pI5.9 and 5.7 respectively, could be obtained free of cross-contamination by isoelectric focusing and had similar specific activities. Although the T isoenzyme could be a catabolic product of the M or the L form, it could also be a unique tumour product, because it was not detected in extracts of normal rat tissues.     

Studies of esterase 6 in Drosophila melanogaster. XIII. Purification and characterization of the two major isozymes

Esterase-6 (EST 6; carboxylic-ester hydrolase; EC 3.1.1.1) from Drosophila melanogaster was purified to homogenity. Purified enzyme occurs as two closely moving isozymes, slow (EST 6S) and fast (EST 6F), on native polyacrylamide gel electrophoresis. Except for slight differences in their mobility, the two isozymes share similar molecular and catalytic properties. Both isozymes are glycoproteins and have an apparent molecular weight of 62,000 to 65,000 as judged by analytical gel filtration and sodium dodecyl sulfate (SDS) electrophoresis. They have identical mobility on SDS-polyacrylamide gels and an isoelectric point of 4.5. Each isozyme has a single active catalytic site as confirmed by titration with 0,0-diethyl-p-nitrophenyl phosphate (Paraoxon). We conclude that EST 6 is a monomeric enzyme. The amino acid composition of the two isozymes is very similar and both variants lack half-cystine residues. The low pI of the enzyme is due in part to a relatively high proportion of glutamic and aspartic amino acid residues. Characterization of the kinetic parameters of the isozymes using beta-naphthyl and p-nitrophenyl esters revealed no statistically significant differences in catalytic efficiency. There is, however, a suggestion that the two isozymes may differ in their substrate specificity.     

A new method for the detection of enolase activity on polyacrylamide gels

A method for the qualitative and quantitative estimation of enolase on polyacrylamide gels after electrophoresis or isoelectricfocusing is described. The procedure is based upon the acid hydrolysis of the primary reaction product, phosphoenolpyruvate, to pyruvate which then is detected as its phenylhydrazone. The method has been successfully applied to crude and homogeneous preparations of enolase from yeast, the free-living nematode, Turbatrix aceti, and rat liver and muscle. Enolase levels as low as 0.2 unit (approximately 0.2 μg) can be readily detected. Though there is an inherent problem of diffusion in the postincubation coupling technique, isozymes can be accurately assayed on gels when separated to the extent of 1 cm.     

Characterization of isoperoxidase activity in Arachis hypogaea cultivars

Isoperoxidases (both anodic and cathodic) from individual seeds of several peanut cultivars (Arachis hypogaea), after ammonium sulfate precipitation of the major storage protein, arachin, from the extracts were characterized by polyacrylamide gel electrophoresis with respect to seed development, maturity and germination and the geographic areas where grown. All cultivars had a major cathodic isozyme near the origin of the gels. Much anodic intra- and inter-varietal isozyme polymorphism was observed in mature seeds collected from different geographic areas. These polymorphic isozymes were consistently present during the earlier stages of seed development (the activities decreased quantitatively and became variable during the later stages of maturation), and most were observed in each peanut upon germination, the latter showing quantitative increases in activity.     

Studies on the Molecular Weights of Esterase and Peroxidase Isozymes of Maize

The molecular weights of esterase and peroxidase isozymes of maize seedlings were directly determined by improved polyacrylamide gradient gel electrophoresis. The different isozyme bands developed in polyacrylamide slab gel electrophoresis (uniform gel) were identified in polyacrylamide gradient gel electrophoresis by means of isozyme variants. The molecular weights of esterase isozymes E1, E2, E3F, E3S, a, b, c, named according to isozyme patterns in uniform gel, are <20000, 35200, 33000, 38500, 29900, 28500, 34000 doltons respectively. The molecular weights of peroxidase isozymes PX4F and PX4S are 131000 and 149000 doltons respectively. According to the band location in uniform gel and in gradient gel, some biochemical properties of the isozyme bands and relationships between the isozyme bands were analyzed. The possible errors in the determination of smaller molecular weight isozymes are discussed.     

Comparison of brain ribonucleases of rabbit,guinea pig,rat, mouse and gerbil

Summary The brain ribonucleases of rabbit, guinea pig, rat, mouse and gerbil were investigated by histochemical and biochemical methods. For the localization, the ribonucleases were electrophoretically transferred from cryostat sections to polyacrylamide gels. Elevated ribonuclease activities were found in the cortex, the basal ganglia, the hippocampal formation and the ventricles, whereas the corpus callosum and the internal capsule exhibited lower activities. The total RNA degrading activities of the brain extracts of the different species varied in a wide range. However, a pre-requisite for the measurement of acid soluble degradation products in the test system was the inactivation of endogencous ribonuclease inhibitors, present in all extracts. Molecular weight analysis by means of SDS-polyacrylamide gel electrophoresis revealed a characteristic set of ribonucleases for each species, consisting of enzymes with different pH-optima.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday