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2.
Nobuyuki Kanzawa Osamu Numata Yoshio Watanabe Koscak Maruyama 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1996,115(4):547-551
Myosin was partially purified from ciliated protozoan Tetrahymena pyriformis. Tetrahymena myosin has a fibrous tail with two globular heads at one end and contains 220-kDa heavy chains. The tail length of the molecule (200 nm) is longer than that of myosins from other animals (approximately 160 nm). A sample after HPLC column chromatography containing 220-kDa peptide showed a myosin-specific K+-/NH4+-EDTA-ATPase activity. Polyclonal anti-crayfish myosin heavy chain antibody reacted with Tetrahymena 220-kDa myosin heavy chain, and monoclonal anti-pan myosin antibody reacted with Tetrahymena 180-kDa peptide. The isolated 180-kDa peptide was identified as a clathrin heavy chain. 相似文献
3.
Vivianne T. Nachmias 《The Journal of cell biology》1972,52(3):648-663
Myosin has been separated from Physarum polycephalum actomyosin in confirmation of the results of Hatano and Tazawa. In an intermediate step, myosin-enriched actomyosin has also been obtained. The mean yield of free myosin was 4.4 mg from 100 g of mold. It was obtained as water-clear solutions at µ = 0.055 with calcium ATPase activity of up to 0.5 µM Pi/min per mg. Negatively stained preparations were examined by electron microscopy. Physarum myosin in 0.5 M KCl interacted with actin from rabbit skeletal muscle to form polarized arrowhead complexes similar to but less regular than those of natural actomyosin from muscle or myosin-enriched Physarum actomyosin. The Physarum myosin-enriched actomyosin at low ionic strength displayed evidence of head-to-tail and tail-to-tail aggregation attributable to the myosin component. Yet Physarum myosin alone did not produce detectable filaments at µ = 0.055 at pH 7, 6.5, or 5.8, nor when dialyzed against 0.01 M ammonium acetate, nor when the dielectric constant of the medium was reduced. However, aggregation approaching the extent of ‘thick filaments’ up to 0.3 µ long was found in some preparations of myosin-enriched actomyosin put into solutions containing adenosine triphosphate. Myosin alone in such solutions did not form filaments. The results are compatible with the idea that head-to-tail aggregations are favored by actin-myosin interactions in Physarum, possibly due to alignment of the extended or tail portions of this myosin molecule. 相似文献
4.
The phosphorylation of synthetic peptides derived from the NH2-terminal sequence of smooth-muscle myosin was studied with purified protein kinase C. The protein kinase C phosphorylation domain included both serine residues and threonine residues in the sequence SSKRAKAKTTKKR(G), denoted myosin light chain (1–13) (MLC(1–13)). Kinetic analysis of MLC(1–13) and truncated peptides derived from the parent peptide established that removal of the serine residues had little effect on protein kinase C reactivity. MLC(1–13) had a V/K of 2.4 min−1·mg−1, whereas the V/K of MLC(3–13) was 3.0 min−1·mg−1. Removal of Lys-3 resulted in a 50% decrease in V/K which was attributable to a 50% decrease in apparent Vmax. Arg-4 was established as a significant protein kinase C specificity determinant, since the apparent Km increased 7-fold and the Vmax decreased 3-fold when the parent peptide was truncated at that residue. All peptides studied required calcium and lipid effectors for full activity with protein kinase C, indicating that they are Class C substrates as defined by Bazzi and Nelsestuen (Biochemistry 26 (1987) 5002) for protein kinase C. Other protein kinases, including cyclic AMP- and cyclic GMP-dependent protein kinase, S6/H4 kinase, myosin light-chain kinase and calcium/calmodulin-dependent kinase II, had little or no activity with these peptides. In studies on the purification of lymphosarcoma protein kinase C by several chromatographic procedures, the results showed that the myosin light-chain can provide convenient and well-characterized substrates for purification and mechanistic studies of protein kinase C biochemistry. 相似文献
5.
Differential binding of contractile proteins from skeletal muscle to Cibacron Blue F3GA-Sepharose affinity columns provides the basis for a new purification technique. Myosin subfragments bind at low ionic strength and are eluted by high salt (e.g., 1.5 m NaCl). Myosin light chain 2 also binds at low ionic strength, whereas light chain 1 is only partially retarded and light chain 3 does not bind. Myosin's marginal solubility in the low-salt buffers required for binding renders it unsuitable for Blue Sepharose chromatography. Neither G-actin nor F-actin bind. Crude preparations of myosin subfragment-1 or light chains undergo significant purification upon Blue Sepharose chromatography. Nee free chromophore inhibits the ATPase activities of myosin and actomyosin at micromolar dye concentrations, whereas the binding of subfragment-1 to actin (in myofibrils) and the tension of glycerinated fibers are inhibited at millimolar dye concentrations. The dye binds at multiple sites on myosin, and inhibits its actomyosin ATPase both competitively and uncompetitively. 相似文献
6.
Ouabain binding was studied in isolated adult dog heart myocytes. The binding was correlated with the inhibition of K+-activated para-nitrophenylphosphatase (K+-PNPPase) activity and the beating response. It was shown that: (i) the specific binding was dependent upon Mg2+ and was inhibited by K+; (ii) the maximal binding capacity (Bmax) was 7.4 × 105 ouabain molecules per cell, or 410 pmol ouabain/K+-PNPPase unit (μmol/min); (iii) in the presence of Mg2+ (5 mm), there were two components in the Scatchard plot, i.e., a high-affinity component with a Kd value of 5.6 × 10?8m and a low-affinity component with a Kd value of 6.7 × 10?7m; (iv) the Hill coefficient (n′) for ouabain binding was 0.72 with a S0.5 value of 7.1 × 10?7m; these values were compatible with the values obtained from studies of K+-PNPPase inhibition by ouabain (n′ = 0.55, S0.5 = 3.6 × 10?7 m) and remained unchanged in the presence of physiological concentrations of Na+ plus K+; (v) in the presence of Mg2+ and K+, the high-affinity component tended to conform to the low-affinity component with an apparent decrease in Bmax; (vi) in the presence of Mg2+ and para-nitrophenylphosphate, the low-affinity component was changed to the high-affinity component with no change in Bmax; (vii) the dissociation rate of the labeled ouabain in the highly dilute medium was not altered in the presence of excess amounts of unlabeled ligand; this eliminated the possibility that the apparent negative cooperativity was due to a site-to-site interaction between receptors; (viii) ouabain increased the number of beating cells and the frequency of beating. Based on these findings, it is concluded that: (i) isolated myocytes possess functional receptors for ouabain; (ii) the binding of ouabain is associated with its inhibition of K+-PNPPase activity; (iii) ouabain receptors in isolated myocytes are of one class with at least two interconvertible conformational states. 相似文献
7.
Myosin has two heads, each of which can interact with actin and ATP. We have investigated the possibility that co-operative interactions occur between the heads by measuring the force generated by single-headed myosin in reconstituted actomyosin threads. Myofibrils were digested with papain, actomyosin was extracted from the myofibrils, and one-headed myosin was purified by cycles of sedimentation with actin. The one-headed myosin was approximately 90 to 95% pure as determined by densitometer scans of polyacrylamide gels run in 20 mm-PP1 (impurities consisted of 1 to 5% of myosin and 1 to 5% myosin rod). The ATPase activity per mole of single-headed myosin was one half that of myosin under conditions where the activity was activated by Ca2+, K+ or actin. One-headed myosin could also participate in superprecipitation, although with a rate that was at least one order of magnitude slower than that for myosin. Myosin or one-headed myosin was mixed with actin, threads were formed via extrusion into low ionic strength, and the isometric forces and isotonic velocities generated by the threads were measured. The ratio of the isometric tension produced per head by the one-headed myosin to the isometric tension produced per head by myosin was 1·0 ± 0·1. The maximum velocity of thread contraction for the one-headed myosin was also not different from the control myosin. Thus, the absence of one head does not appear to impair the generation of force or motion by the remaining head. 相似文献
8.
Myosin reacted with N-ethylmaleimide in the presence of ADP lost its ability to be activated by actin. Subfragment 1 behaved similarly. About 2 moles of N-ethylmaleimide per mole of Subfragment 1 were required to eliminate actin activation of the Mg2+-ATPase activity. At the point at which actin activation was lost the K+-EDTA-ATPase activity was also lost, but the Ca2+-activated ATPase activity was increased. Kinetic measurements indicated that the labelling with N-ethylmaleimide in the presence of ADP reduced V (the ATPase activity at infinite actin concentration) but did not effect Kapp (which is related to the dissociation constant of the actin-Subfragment 1 complex). The Mg2+-activated activity of the reacted myosin alone remained unaltered and the ability to bind actin was retained. We propose that the N-ethylmaleimide labelling blocked the actin activation by preventing the accelerated release of hydrolysis products from the myosin. 相似文献
9.
Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles 总被引:4,自引:3,他引:1
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Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2′, 5′-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GRA and GRB). Purified GRA had a specific activity of 1.81 microkatals per milligram of protein and GRB had a specific activity of 6.08 microkatals per milligram of protein. GRA accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GRB accounted for 83%. The native molecular mass for GRA was 103 to 104 kilodaltons and for GRB was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GRA and 57 kilodaltons for GRB. The pH optimum for GRA was 7.25 to 7.75 and for GRB was 7.25. At 25°C the Km for GSSG was 15.3 and 39.8 micromolar for GRA and GRB, respectively. For NADPH, the Km was 3.7 and 8.8 micromolar for GRA and GRB, respectively. Antibody produced from purified GRB was reactive with both native and denatured GRB, but was cross-reactive with only native GRA. 相似文献
10.
Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems 总被引:4,自引:2,他引:2
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Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (Km = 12 micromolar) and coniferaldehyde (Km = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical. 相似文献
11.
《Neurochemistry international》1987,11(1):69-75
l-[3H]Glutamate binding to synaptic plasma membranes from rat cerebral cortices was carried out at 2–4°C in 50 mM Tris-acetate buffer (pH 7.4) using a microfuge centrifugation method. Binding was increased by repeated freezing-thawing and washing in either crude or partially purified synaptic membranes. Scatchard analysis showed a single binding site (dissociation constant, KD = 697 nM; maximal binding capacity, Bmax = 7.5 pmol/mg protein) in four times distilled water washed crude synaptic membrane. After six times freezing-thawing and washing, a new high affinity site (KD1 = 26 nM, Bmax1 = 1.8 pmol/mg protein) appeared and the number of low affinity site was increased with no apparent change in affinity (KD2 = 662 nM, Bmax2 = 10.5 pmol/mg protein). l-[3H]Glutamate binding was inhibited by acidic amino acid analogues that interact with N-methyl-d-aspartate- and quisqualate-sensitive sites of glutamate receptors. Binding was marginally inhibited by kainate and l-2-amino-4-phosphonobutyrate. These results indicate that repeatedly frozen-thawed and washed synaptic plasma membrane is suitable for studying the subtypes and regulation of glutamate receptors. 相似文献
12.
The sedimentation equilibrium in concentrated solutions of hemoglobin and myoglobin has been measured. The ratio of the apparent molecular weight of hemoglobin to that of myoglobin. R. is found to obey the empirical relation R(c) = 3.8–4.25 × 10?2c+6.44 × 10?4c2 ? 2.21 × 10?5c3, where c is the protein concentration in g/dl. for c??40 g/dl. A theoretical relation for the dependence of R upon c in the absence of protein self-association is presented. This relation cannot be fitted to the experimental results, and the discrepancy is attributed to self-association of myoglobin. 相似文献
13.
《Experimental mycology》1989,13(1):13-19
The purification and kinetic characterization of uridine phosphorylase from Dictyostelium discoideum are described. Matrex Green A, a dye-affinity chromatography gel, was used for the purification. The enzyme was specifically eluted from the dye bead matrix with the use of its substrate, uridine, resulting in a purification of 70- to 2000-fold. The enzyme preparation exhibited stoichiometry. For nucleoside phosphorolysis, the Km values for phosphate and uridine were 0.42 and 0.24 mm, respectively, and the Ki for phosphate was 3.0 mm. For nucleoside synthesis, the Km values for uracil and ribose 1-phosphate were 0.06 and 0.14 mm, respectively, and the Ki for ribose 1-phosphate was 0.05 mm. An ordered sequential bi:bi mechanism is proposed based on product inhibition studies. 相似文献
14.
Separation and characterization of four hexose kinases from developing maize kernels 总被引:2,自引:2,他引:0
Doehlert DC 《Plant physiology》1989,89(4):1042-1048
Four forms of hexose kinase activity from developing maize (Zea mays L.) kernels have been separated by ammonium sulfate precipitation, gel filtration chromatography, blue-agarose chromatography, and ion exchange chromatography. Two of these hexose kinases utilized d-glucose most effectively and are classified as glucokinases (EC 2.7.1.2). The other two hexose kinases utilized only d-fructose and are classified as fructokinases (EC 2.7.1.4). All hexose kinases analyzed had broad pH optima between 7.5 and 9.5 with optimal activity at pH 8.5. The two glucokinases differed in substrate affinities. One form had low Km values [Km(glucose) = 117 micromolar, Km(ATP) = 66 micromolar] whereas the other form had much higher Km values [Km(glucose) = 750 micromolar, Km(ATP) = 182 micromolar]. Both fructokinases had similar substrate saturation responses. The Km(fructose) was about 130 micromolar and the Km(ATP) was about 700 micromolar. Both exhibited uncompetitive substrate inhibition by fructose [Ki(fructose) = 1.40 to 2.00 millimolar]. ADP inhibited all four hexose kinase activities, whereas sugar phosphates had little effect on their activities. The data suggest that substrate concentrations are an important factor controlling hexose kinase activity in situ. 相似文献
15.
Production of Beauvericin, Moniliformin, Fusaproliferin, and Fumonisins B1, B2, and B3 by Fifteen Ex-Type Strains of Fusarium Species
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Fifteen Fusarium species were analyzed by high-performance liquid chromatography for the production of six mycotoxins in corn grits cultures. Production of mycotoxins ranged from 66 to 2,500 μg/kg for fumonisin B1, 0.6 to 1,500 μg/g for moniliformin, 2.2 to 720 μg/g for beauvericin, and 12 to 130 μg/g for fusaproliferin. Fumonisin B2 (360 μg/kg) was produced by two species, fumonisin B3 was not detected in any of the 15 species examined, and Fusarium bulbicola produced none of the six mycotoxins that we analyzed. 相似文献
16.
The performance of two solid phase extraction (SPE) purification procedures, used in the determination of fumonlsin B1 (FB1), B2 (FB2) and B3 (FB2) In corn, was evaluated using both thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Fewer interferences were observed In extracts prepared using the strong anion exchange (SAX) media, in contrast to those purified on C18 media, where on occasions, visual discernment of the TLC bands was hampered by the presence of interfering compounds. Precipitate formation, resulting In the blocking of SPE cartridges was also encountered when using the C18 procedure. HPLC analyses of extracts prepared by both media indicated that they gave comparable fumonlsin recoveries from naturally contaminated corn samples. The results suggest that the C18 procedure, originally developed for the TLC analyses of FB1 in mixed feeds, may also be applied to the determination of FB2 and FB2. However, where TLC is used quantitatively for fumonlsin levels <1 μg/g, purification of sample extracts on SAX media is recommended. 相似文献
17.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2013,7(1):45-51
The effects of activation and inhibition of serotonin receptors by serotonin (5-HT) and mianserin on the specific nonselective α1-antagonist [3H]prazosine binding in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction of α1-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and the binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: K d =1.85 ± 0.16 nM, B max = 31.1 ± 0.3 fmol/mg protein, n = 2. In case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K d1 = 0.61 ± 0.04, K d2 = 3.82 ± 0.15 nM, B m1 = 6.6 ± 0.7, B m2 = 25.6 ± 0.4 fmol/mg protein, n = 2. The sensitivity of the high-affinity pool increased threefold and the sensitivity of the low-affinity pool decreased twofold as compared to the control. The value of maximal reaction (B max) did not change. In the case of inhibition of 5HT-receptors by mianserin, radioactive ligand is bound to α1-adrenoceptors according to the same model as in the control conditions. The affinity of α1-adrenoceptors to [3H]prazosine decreases twofold and the concentration increases (K d = 3.97 ± 0.12 nM, B max = 40.0 ± 0.5 fmol/mg protein). The data suggest that α1-adrenoceptors in rat cerebral cortex exist as a dimer. The modulatory effects of serotonin and mianserin on the specific binding of [3H]prazosine to α1-adrenoceptors was detected, manifesting itself as changes in the binding parameters and in the general character of ligand-receptor interactions. 相似文献
18.
《Inorganica chimica acta》1986,113(2):181-186
Axial ligation constants (log KB) of bases for [5, 10,15,20-tetraphenylporphyrinato(2-)]cobalt ([CoII (tpp)]) are reported. The log KB values of pyridine derivatives except for 4-cyanopyridine show a good linear relationship in a plot of log KB vs. pKa of the axial ligand. 4-Cyanopyridine gives a larger log KB than expected. The log KB value of 1-methylimidazole for [CoII(tpp)] is almost the same as that for tetrakis(p-methoxyphenyl)porphyrinatocobalt(II) ([Co((p-CH3O)tpp)]), althoug the other log KB values for [CoII(tpp)] are always slightly larger than those for [Co((p-CH3O)tpp)]. These results are discussed on the basis of the σ- and π-bonding abilities of the bases, and the solvent effects on the log KB values. The lower base affinities of cobalt(II) capped porphyrins are also discussed. 相似文献
19.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2013,7(4):280-285
The influence of activation and inhibition of serotonin receptors by serotonin (5HT) and miancerin on binding of specific nonselective α2-antagonist [3H]RX821002 in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction for α2-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of the [3H]RX821002 binding to α2-adrenoceptors were as follows: K d = 1.57 ± 0.276 nM, B max = 7.24 ± 1.63 fmol/mg protein, n = 2. In the case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K d1 = 0.82 ± 0.06; K d2 = 2.65 ± 0.22 nM; B m1 = 1.65 ± 0.23; B m2 = 4.20 ± 0.11 fmol/mg protein; n = 2. The affinity of high-affinity receptors increased twofold and the affininty of low-affinity receptors decreased by 69% as compared to control values. The concentration of high-affinity receptors decreased 4.4-fold, and of low-affinity, 1.7-fold. The value of maximal reaction (B max) decreased by 20%. In the case of miancerin-induced inhibition of 5HT-receptors the character of ligand binding also changed; two pools of receptors were detected with the following parameters: K d1 = 0.48 ± 0.09; K d2 = 3.79 ± 0.71 nM; B 1 = 0.63 ± 0.17; B 2 = 4.75 ± 0.21 fmol/mg protein; n = 2. The affinity of high-affinity receptors pool increased by 70% and that of low-affinity receptors decreased by 76% as compared to control values. The concentration of active high-affinity and low-affinity α2-adrenoceptors decreased by 70% and 141%, respectively. The total amount of the receptors (B max) decreased by 26%. The data suggest that α2-adrenoceptors in rat cerebral cortex exist as dimers. Modulatory effects of serotonin and miancerin on specific antagonist binding to α2-adrenoceptors may be accomplished by altering the character and binding parameters of the nonselective α2-antagonist [3H]RX821002. 相似文献
20.
We have characterized the thromboxane (TX) A2/prostaglandin (PG) H2 receptor in glomeruli isolated from the rat using the agonist radioligand [125I]-BPO. Binding of [125]-BOP was highly specific, stereoselective, and to a single class of high affinity binding sites (Kd = 1/16 ± 0.22 nM and Bmax = 348 ± 32 fol/mg protein; n = 6). Binding of [125I]-BOP was competed for by the agonist ONO11113 (Kd = 50.8 ± 8.0 nM; n = 4) and the antagonists SQ29548 (Kd = 15.8 ± 1.0 nM; n = 3), L657925 (Kd = 12.1 ± 2.2 nM; n = 3) and L65796 (Kd = 1642 ± 135 nM; n = 3). I-BOP also produced a TXA2/PGH2 receptor-mediated rise in [CA2+]i in isolated glomeruli In adriamycin-induced nephrotic syndrome in the rat, the development of proteinuria is reported to be dependent on increased renal TXA2 production. We therefore examined whether or not changes in glomerular TXA2/PGH2 receptors occur between control and nephrotic rats. No changes in expression of affinity of either glomerular or platelet TXA2/PGH2 receptors were observed. Kd and Bmax values for isolated isolated glomeruli were 1.45 ± 0.24 nM and 406 ± 72 fmol/gm for controls and 1.22 ± 0.25 nM and 321 ± 62 fmol/gm for nephrotic rats (n = 6). 相似文献