Stabilization of activity of oxidoreductases by their immobilization onto special functionalized glass and novel aminocellulose film using different coupling reagents

Glucose oxidase (GOD), horseradish peroxidase (HRP), and lactate oxidase (LOD) were covalently immobilized on special NH(2)-functionalized glass and on a novel NH(2)-cellulose film via 13 different coupling reagents. The properties of these immobilized enzymes, such as activity, storage stability, and thermostability, are strongly dependent on the coupling reagent. For example, GOD immobilized by cyanuric chloride on the NH(2)-cellulose film loses approximately half of its immobilized activity after 30 days of storage at 4 degrees C or after treatment at 65 degrees C for 30 min. In contrast, GOD immobilized by L-ascorbic acid onto the same NH(2)-cellulose film retains 90% of its initial activity after 1 year of storage at 4 degrees C and 92% after heat treatment at 65 degrees C for 30 min. Unlike GOD, in the case of LOD only immobilization on special NH(2)-functionalized glass, e.g., via cyanuric chloride, led to a stabilization of the enzyme activity in comparison to the native enzyme. The operational stability of immobilized HRP was up to 40 times higher than that of the native enzyme if coupling to the new NH(2)-cellulose film led to an amide or sulfonamide bond. Regarding the kinetics of the immobilized enzymes, the coupling reagent plays a minor role for the enzyme substrate affinity, which is characterized by the apparent Michaelis constant (K(M,app)). The NH(2)-functionalized support material as well as the immobilized density of the protein and/or immobilized activity has a strong influence on the K(M,app) value. In all cases, K(M,app) decreases with increasing immobilized enzyme protein density and particularly drastically for GOD.     

β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis

The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.     

Multiple Defects in the Activity of Adenylate Cyclase from the Drosophila Memory Mutant Rutabaga

Adenylate cyclase in homogenates of Drosophila melanogaster is heterogeneous with respect to its affinity toward MgATP and its subcellular distribution. Km values for MgATP range, under similar assay conditions, from approximately 10(-5) M to approximately 10(-3) M, depending on the body region and on the subcellular localization of the enzyme. The majority of the enzyme in whole-body preparations is particulate, but various body regions differ in the relative proportion of the soluble enzyme. The memory mutant rutabaga lacks up to 35% of the total particulate activity. Even ligands that stimulate directly the catalytic subunit are incapable of bringing the activity of the mutant's enzyme to normal levels. The defect is differentially pronounced in various body parts and is associated with an altered responsiveness of the enzyme to Mg2+, to Ca2+, and to forskolin. It is suggested that rutabaga is lesioned in a subpopulation, or a functional state, of adenylate cyclase, which may play a role in memory formation.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

Guanylate cyclase of isolated bovine retinal rod axonemes.

The guanylate cyclase activity of axoneme--basal apparatus complexes isolated from bovine retinal rods has been investigated. The Mg2+ and Mn2+ complexes of GTP4- serve as substrates. Binding of an additional mole of Mg2+ or Mn2+ per mole of enzyme is required. Among cations which are ineffective are Ca2+, Ni2+, Fe2+, Fe3+, Zn2+, and Co2+. The kinetics are consistent with a mechanism in which binding of Mg2+ or Mn2+ to the enzyme must precede binding of MgGTP or MnGTP. The apparent dissociation constants of the Mg--enzyme complex and the Mn--enzyme complex are 9.5 x 10(-4) and 1.1 x 10(-4) M, respectively. The apparent dissociation constants for binding of MgGTP and MnGTP to the complex of the enzyme with the same metal are 7.9 x 10(-4) and 1.4 x 10(-4) M, respectively. The cyclase activity is maximal and independent of pH between pH 7 and 9. KCl and NaCl are stimulatory, especially at suboptimal concentrations of Mg2+ or Mn2+. Ca2+ and high concentrations of Mg2+ and Mn2+ are inhibitory. Ca2+ inhibition appears to require the binding of 2 mol of Ca2+ per mol of enzyme. The dissociation constant of the Ca2--enzyme complex is estimated to be 1.4 x 10(-6) M2. The axoneme--basal apparatus preparations contain adenylate cyclase activity whose magnitude is 1--10% that of the guanylate cyclase activity.     

Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost.

Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 3.2.1.52). Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M. The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5. Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.     

Purification and characterization of a carboxylesterase from rabbit liver lysosomes

Carboxylesterase [EC 3.1.1.1] was purified from rabbit liver lysosomes by means of detergent solubilization, and by hydroxyapatite, phenyl-Sepharose and chromatofocusing column chromatographies. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 58,000. This enzyme was eluted at an isoelectric point of approximately 5.8 by chromatofocusing, and exhibited a broad pH optimum of between 6.0 and 9.0. The enzyme hydrolyzed 4-methylumbelliferyl esters of saturated fatty acids (C2-C12), and it also hydrolyzed p-nitrophenylacetate, methyl butyrate, and tributyrin, but not acetanilide. Its activity was completely inhibited by diisopropyl-fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10(-4) M, but was not affected by eserine, or by alpha- or beta-naphthyl acetate at 10(-3) M. Various metal ions (Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Zn2+, Ni2+) at 10(-3) M also had no effect on the enzyme activity.     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

Interaction of AMP-aminohydrolase with myosin and its subfragments.

We have shown that purified rabbit skeletal muscle AMP-aminohydrolase binds to rabbit muscle myosin, heavy meromyosin, and Subfragment 2 but does not bind to light meromyosin nor to Subfragment 1. The dissociation constant for binding to myosin was determined to be 0.14 muM. A new sedimentation boundary, presumably reflecting formation of a complex between AMP-aminohydrolase and heavy meromyosin or Subfragment 2, can be observed using the analytical ultracentrifuge. Binding of AMP-aminohydrolase to myosin, heavy meromyosin, or Subfragment 2 is abolished by phosphate (less than 10 mM), an inhibitor of AMP-aminohydrolase. No other rabbit muscle enzyme tested showed any interaction with myosin under the same conditions and there was no indication of complex formation between AMP-aminohydrolase and phosphofructokinase or phosphocreatine kinase in the analytical ultracentrifuge.     

Lipoamide dehyrogenase immobilized on porous glass.

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) isolate from pig heart and Escherichia coli was covalently coupled by both diazonium and amide bonds to controlled pore glass beads (96% silica). When the enzyme was immobilized in the presence of NAD+, the enzyme no longer exhibited its normal requirement for NAD+ for full activity. If the immobilized enzyme was then treated with NADase, the requirement for NAD+ was restored. Enzyme immobilized in the absence of NAD+ exhibited normal NAD+ dependence both prior to an after NADase treatment. These results are discussed in terms of co-immobilization of NAD+ at or near the allosteric site of the enzyme.     

Differentiation of fluorides-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase cy calcium ions, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid and Triton X-100.

Beef brain cortex adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity is 84--88% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the absence of F- but only 50--60% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the presence of F-. In either case, further increase in EGTA concentration did not alter the degree of inhibition. The inhibition can be completely reversed in both cases by addition of 3 - 10(-5) M Ca2+, (yielding a [free Ca2+] of approximately 2 - 10(-6) M) and 5 - 10(-5) M Mn2+ or Co2+ and partially by 5 - 10(-5) M Sr2+ but not by addition of 5 - 10(-5) M Ba2+, Zn2+, Ni2+ or Fe2+. A [free Ca2+] of 7.2 - 10(-5) M markedly inhibited cyclase activity in the presence of F-. Solubilization by 1.8% Triton X-100 resulted in an enzyme preparation no longer stimulated by NaF and 100% inhibited by the addition of 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid either in the absence or presence of NaF. However, in contrast to ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-TETRAACETIC ACID, EDTA had no measurable effect on adenylate cyclase either in the presence or absence of NaF and ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid did not affect ATPase or phosphodiesterase activities. The data is rationalized by the postulation of two independent enzyme components in brain cortex: one component is about six-fold activated by NaF and the NaF effect is enhanced by low concentrations of Ca2+ and Mg2+. A second component is totally Ca2+ dependent and inhibited by high concentrations of F-. Mn2+, Co2+ and Sr2+ appear to be in vitro Ca2+ substitutes for both enzyme systems. On this basis, Triton X-100 treatment results in about a three-fold increase in specific activity of the Ca2+ dependent cyclase component but a complete abolition of the NaF stimulated component.     

Purification of a moderate thermotolerant Bacillus coagulans BTS1 lipase and its properties in a hydro-gel system

An alkaline thermotolerant lipase of Bacillus coagulans BTS1 was successively purified by ammonium sulfate precipitation and DEAE anion exchange chromatography. The purified lipase immobilized in alginate beads showed an optimal activity at pH 7.5 and 55 degrees C. A pH of 5.0 or 10.0 completely quenched the activity of immobilized lipase. The alginate-bound lipase retained its activity following exposure to most of the organic solvents including amines, alkanes and alcohols. Chloride salt of Al3+, Co2+, Mg2+ and NH4+ modulated the lipase activity of alginate-immobilized enzyme. The alginate entrapped lipase showed a preferentially high activity towards p-nitrophenyl palmitate (C: 16) and activity of matrix increased following exposure to SDS. Moreover, the immobilized lipase retained more than 50% of its activity after 3rd cycle of reuse.     

游离及固定化果糖基转移酶部分酶学性质的比较研究

 从诱变、筛选的米曲霉GX0 0 10菌株所产生的果糖基转移酶 ,经过纯化和固定化操作分别制备游离酶和固定化酶 ,对两者的酶学性质进行了比较研究 .结果表明 ,两者在蔗糖转化为蔗果低聚糖的酶促反应中 ,最适pH为 5 5,在pH5 0～ 7 5之间酶活性相对稳定 .游离酶和固定化酶的适宜温度范围分别是 4 5～ 52℃和 4 0～ 55℃ .在 55℃保温 60min ,酶活性保存率分别是 61 6%和 87 5% .固定化酶的热稳定性提高 .0 1mmol LHg2 +和 1mmol LAg+能完全抑制游离酶的活性 ,但只能部分抑制固定化酶的活性 ,1mmol L的Ti2 +能完全抑制两者的活性 .以蔗糖为底物时 ,游离酶的米氏常数Km=2 15mmol L ,而固定化酶Km =386mmol L .游离酶只能使用一次 ,固定化酶反复使用 54次后 ,剩余活力为 55 2 % .用 55% (W V)蔗糖溶液与固定化酶在pH5 0 ,4 6℃下作用 12h ,可获得61 5% (总低聚糖 总糖 )产物 ,其中蔗果五糖含量达到 7 2 % .     

尼龙固定化木瓜蛋白酶及其应用研究

 尼龙经CaCl_2和H_2O的甲醇溶液处理,稀HCl水解用戊二醛交联以制备固定化木瓜蛋白酶。在溶液酶浓度为1mg/mL pH7.5—8.0、4—15℃条件下固定3h,活力回收42.5％,相对活力46％,偶联效率52％,半衰期72天。溶液酶Km值和固定化酶K_m~(aPP)值(底物酪蛋白W/V,％)分别为0.28％和0.35％。溶液酶和固定化酶分别在pH6.5和pH8.0以下活力稳定;最适pH分别为7.0和8.0;在65℃处理30min活力分别为原有活力的89％和66％。当酪蛋白浓度为1.5％和2.5％以上活力分别受到抑制。固定化酶在6mol/L脲中连续浸洗5次共6h其活力稳定,仍有原活力的44.4％;用以处理啤酒浊度比对照下降了2-11倍;蛋白质含量下降了55％;冷藏(4℃)120天,无冷混浊发生;同时各项理化指标和风味不变。     

Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 x 10(-5) mmol BCA/m(2), and on graphite it was 1.7 x 10(-3) mmol BCA/m(2) nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4 degrees C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23 degrees C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second-order rate constant K'(Enz) for the hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25 degrees C is 2.6 x 10(2) and 5.6 x 10(2) M(-1)s(-1) respectively. The corresponding K(ENZ) value for the free enzyme is 9.1 x 10(2) M(-1)s(-1). Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pK(a), are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA-active site pK(a) values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.     

Immobilization of rat lung soluble guanylate cyclase on alkyl-agarose gels.

The soluble guanylate cyclase from rat lung was immobilized by absorption rather than covalent attachment on hexyl-, octyl-, or decyl-agarose. The enzyme retained activity after being bound to these matrices and could be compared to the soluble, mobile form of the enzyme. Compared to the soluble enzyme, the immobilized guanylate cyclase had a lower apparent maximal velocity and a higher apparent Km for MeGTP in the presence of Mg2+, Ca2+, or Mn2+. The apparent maximum velocity was reduced to the same extent by hexyl-, octyl-, or decyl-agarose, but the reduction in activity was greater with Mg2+ than with Ca2+ or Mn2+. Both the soluble and immobilized guanylate cyclase displayed concave downward patterns on double reciprocal polots as a function of Mn2+, and Ca2+ caused apparent activation of either form of the enzyme. MnATP appeared to be a linear competitive inhibitor with respect to MnGTP for both forms of the enzymes but the ki was 3 micron for the soluble form and 30 micron for the immobilized form. These results demonstrate that the soluble form of guanylate cyclase from rat lung retains many of its basic properties after being immobilized on a hydrophobic matrix; however, rather pronounced decreases in the maximum velocity and increases in the apparent Michaelis constant for MeGTP, particularly for MgGTP, are observed upon immobilization.     

Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.     

Kinetic studies of sepharose-and CH-sepharose-immobilized dihydrofolate reductase

Dihydrofolate reductase, purified to homogeneity from amethopterin-resistant Lactobacillus casei, was immobilized by coupling to cyanogen bromide-activated Sepharose or carbodiimide-activated CH-Sepharose. Coupling yields were determined by amino acid analysis following the hydrolysis of the gel. Enzyme activity was measured by the conventional spectrophotometric procedure, thus permitting the facile characterization of the immobilized enzyme. The pH optimum of the immobilized enzyme was shifted to 5.8 compared with pH 5.5 for the soluble enzyme. The immobilized enzyme retained greater than 90%of the initial activity over a six-month period and could be reused as many as ten times without loss of activity. As observed with the soluble enzyme, the activity of immobilized enzyme, which was lost on denaturation with 4M guanidine hydrochloride, was recovered rapidly and completely by washing the gel with buffer. The K(m) (app) values for dihydrofolate and NADPH for the immobilized enzyme were increased 15-164-fold over the K(m) values measured for soluble dihydrofolate reductase. Scatchard analysis of the interaction of amethopterin with the immobilized enzyme yielded linear plots and a K(d) (app) value of 0.56 x10(-8)M, and revealed that all of the immobilized enzyme molecules were capable of binding the ligand.     

The influence of lysosomes on glycogen metabolism.

Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6'-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C.     

Variance analysis of Cd2+, Zn2+, and Mn2+ effect on membrane Na+,K(+)-ATPase activity of loach embryos

The evaluation of influences Cd2+, Zn2+, Mn2+ in concentrations 10(-6), 10(-5), 10(-4) M (factor of dose) on the Na+, K(+)-ATP-ase activity in the early period of development (60-330 min.) of loach embryos (time factor) using one- and two-factor analysis of variance has been performed. It has been detected, that the changes of enzyme activity are mainly caused by action of the explored cations and do not depend on time of embryos development. The most influence on activity in the indicated period of embryos development of loach renders Cd2+ in concentration 10(-4) M, relative value of its influence being 95.7% (p = 0.01). Substantial concentration dependence of the Na+, K(+)-ATP-ase activity is exposed to the action of each of cations. The values of the influence of their concentration changes during the studied period of development differ insignificantly for all cations and make 76.2-77.5% (p < 0.01).