ADENYLATE KINASE BOUND TO THE CHROMATOPHORE MEMBRANES OF Rhodobactor spheroides G1C

Transformation of adenylates (AMP, ADP and ATP) by washed chromatophore membranes of Rhodobactor spheroides G1C in the dark and in the light indicated the functions of ATPase (ADP + Pi in equilibrium ATP) and of an adenylate kinase (2ADP in equilibrium AMP + ATP). The activity of adenylate kinase of the chromatophores was not inhibited by AP5A, and persisted even after sonication in the presence of EDTA or CaCl2; the results suggested the presence of an adenylate kinase bound to the chromatophore membrane. In search of the enzyme, the supernatant after sonication of the chromatophores in the presence of EDTA was subjected to a molecular sieve and then to ion-exchange HPLC; a fraction with high specific adenylate kinase activity, containing a very sharp peak at 55 kDa, was isolated. Preliminary characterization indicated that it is different from the well-documented water-soluble 33 kDa adenylate kinase.     

ATP synthesis in the disk membranes of rod outer segments of bovine retina

ATP is synthesized on the disk membrane isolated from rod outer segments of the bovine retina. Together with a slow component which accounted for a constant rate of about 22 nmol ATP/min/mg of protein and which was due to the adenylate kinase activity, a fast component with a maximal activity of about 58 nmol ATP/min/mg of protein was measured at physiological calcium concentrations. This fast activity disappeared in the presence of the Ca(2+) ionophore A23187, was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting that this ATP synthesis is due to the reversal functioning of the Ca(2+)-ATPase previously found on the disk membranes.     

Characterization of thyroidal membrane-bound Mg-adenosinetriphosphatase activated by trypsin or poly-L-lysine.

The Mg-adenosinetriphosphatase (ATPase) in the thyroidal NaI-treated microsome fraction was activated by treatment with basic polyamino acids or trypsin, but not with acidic polyamino acids and basic proteins such as lysozyme and ribonuclease. The enzyme kinetics showed that the activation of trypsin or poly-L-lysine was due to an increase in the maximal velocity of the hydrolyzing reaction without a change in the affinity of the enzyme for its substrate. A break at about 25 degrees C was observed in the Arrhenius plots of Mg-ATPase in the trypsin- or poly-L-lysine treated preparations, but there was no break in the control preparation. These results suggest that the activating effect of trypsin or poly-L-lysine on Mg-ATPase activity in the thyroidal NaI-treated microsome fraction is related to the lipid environment surrounding the enzyme molecule in the thyroid cell membrane.     

Effect of vanadate on gill cilia: switching mechanism in ciliary beat.

Lateral (L) cilia of freshwater mussel (Margaritana margaritifera and Elliptio complanatus) gills can be arrested in one of two unique positions. When treated with 12.5 mM CaCl2 and 10(-5) M A23187 they arrest in a "hands up" position, ie, pointing frontally. When treated with approximately 10 mM vanadate (V) they arrest in a "hands down" position, ie, pointing abfrontally. L-cilia treated with 12.5 mM CaCl2 and 1 mM NaN3 also arrest in a "hands down" position; substitution of 20 mM KCl and 1 mM NaN3 causes cilia to move rapidly and simultaneously to a "hands up" position. The observations suggest that there are two switching mechanisms for activation of active sliding in ciliary beat one at the end of the recovery stroke and the other at the end of the effective stroke; the first is inhibited by calcium and the second by vanadate or azide. This is consistent with a model of ciliary beating where microtubule doublet numbers 1, 2, 3, and 4 are active during the effective stroke while microtubule doublets numbers 6, 7, 8, and 9 are passive, and the converse occurs during the recovery stroke.     

Calcium regulation of cardiac myofibrillar activation: effects of MgATP.

In 2 mM MgATP, 0.08 ionic strength and 1 mM free Mg++ cardiac myofibrils bound 3.5 nmoles Ca/mg protein at maximal ATPase activation. Significant amounts of Ca were also bound to cardiac myosin with these same conditions. By subtraction of this myosin-bound Ca we obtained an estimate of 4 moles Ca bound per mole of myofibrillar troponin at maximal ATPase. We found, however, that Ca activation of myofibrillar ATPase could be estimated assuming that only two of troponin's Ca-binding sites are engaged in regulation of crossbridge activity. Increases in MgMTP from 0.3 to 5.0 mM raised the free Ca, giving half-maximal isomteric tension or ATPase. Although part of this shift is most probably due to changes in the number of rigor (nucleotide-free) actin-myosin linkages, the rightward shift of the free Ca++-activation relation with increase in MgATP from 2 to 5 mM appears to be due to effects of active (nucleotide-containing) actin-myosin linkages.     

UV-ACTION SPECTRUM (254-405 nm) FOR INHIBITION OF A K+ -STIMULATED ADENOSINE TRIPHOSPHATASE FROM THE PLASMA MEMBRANE OF ROSA DAMASCENA

A K ⁺-stimulated ATPase from suspension-cultured rose cells was isolated and subjected to UV radiation. The characteristics of the ATPase resembled those of a plasma-membrane associated enzyme and not those of the mitochondrial enzyme. The ATPase required Mg²⁺ and was further stimulated up to 100% by K⁺. K⁺ stimulation was specific for ATP. The order of stimulation by monovalent cations was K⁺ > Na⁺ > Li⁺. The enzyme had a pH optimum of 6.5 in the presence of 50 mM K⁺. It was almost completely inhibited by diethylstilbestrol and partially inhibited by vanadate. but was not affected by azide or oligomycin. The inhibition of ATPase activity by various fluences of UV indicated that one fraction of the K⁺-stimulated activity was very sensitive to radiation, while another fraction was relatively insensitive. It is possible that UV distinguished between two enzymes. The action spectra for inhibition of both fractions showed maxima at 290 nm and significant but much lower action throughout the near-UV region, resembling spectra in the literature for the inhibition of transport processes in bacteria.     

A high-affinity ATP-binding site on 30S dynein.

The enhancing effect of low concentrations (eg, 8 microM) of bis(4-fluoro-3-nitrophenyl)sulfone (FNS) on 30S dynein ATPase activity is increased when 1 mM dithiothreitol (DTT) is present. The effect of FNS + DTT is optimal at pH 7.5. Activation of the latent ATPase activity of 30S dynein by FNS + DTT is partially prevented by 1--3 microM ATP. Adenylylimidodiphosphate (AMP-PNP) is less effective than ATP, while beta, gamma-methylene-adenosine triphosphase (AMP-PCP), though a much stronger inhibitor of ATPase activity than AMP-PNP, does not protect against enhancement. These results demonstrate the presence of high-affinity ATP-binding site on 30S dynein.     

Rat adipose tissue phosphatidic acid phosphatase: lack of effect of nucleotides on cytosolic enzyme activity

The soluble phosphatidic acid phosphatase from rat adipose tissue was partially purified using ammonium sulfate fractionation and hydroxyapatite chromatography. Administration of ethanol has been found to increase phosphatidic acid phosphatase activity. The enzyme activity has been found to be dependent on magnesium ions with maximal activity at 2-5 mM magnesium. The enzyme displays an apparent pH optimum of 7.0. The activity of the enzyme is not affected by addition of ATP or ADP, in contrast with the results for hepatic phosphatidic acid phosphatase. The results suggest that these two enzymes may be regulated by different mechanisms and that they may thus represent two different types of isoenzyme.     

Purification and characterization of adenosine diphosphatase from human umbilical vessels.

Adenosine diphosphatase (ADPase) activity was solubilized with a non-ionic detergent, Tween 20, from human umbilical vessels and purified to homogeneity by diethylaminoethyl-Sepharose CL-6B, adenosine 5'-monophosphate-Sepharose 4B, and concanavalin A-Sepharose chromatography. The apparent molecular mass was 75 kDa. The purified enzyme hydrolyzed pyrophosphate bonds of nucleoside di- and triphosphates in the presence of calcium ion. It was insensitive to the adenosine triphosphatase (ATPase) inhibitors, oligomycin and ouabain, and sensitive to sodium azide. Therefore, we concluded that the ADPase activity in human umbilical vessels does not derive from ADPase degrading only ADP but from ATP diphosphohydrolase (EC 3.6.1.5). The broad substrate specificity and the sensitivity to various inhibitors and calcium ion are common to ATP diphosphohydrolase from bovine aorta. However, there might exist some structural difference around the active site, because the antiserum raised in rabbit against the bovine aorta enzyme scarcely inhibited the human umbilical enzyme.     

Purification and characterization of acetate kinase from Clostridium thermocellum

Acetate kinase (EC 2.7.2.1), an enzyme involved in the wasteful production of acetate during conversion of cellulose to ethanol by Clostridium thermocellum, was purified 144-fold. The enzyme has an Mr of 84 kD by non-denaturing gradient gel electrophoresis, and an Mr of 46 kD when estimated with a denaturing gel; thus it appears to be a homodimer. Optimum enzyme activity occurs at 50°C and between pH 7.2 and 8.0. Acetate kinase is stable to temperatures up to 60°C, but is completely inactivated at 80°C after two h. The enzyme is stable between pH 7.0 and 9.0 when incubated at 50°C for two h. Optimum acetate kinase activity occurs at a MgCl₂:ATP ratio of 2:1, which indicates an interaction between Mg²⁺ and ATP and that between Mg²⁺ and acetate kinase. Enzyme activity is partially inhibited by KCl, an inorganic salt frequently used in chromatography and fermentation media, losing 60% activity in the presence of 0.2 M KCl. Sigmoidal enzyme kinetics were observed from the velocity plot of acetate kinase when either the acetate (S_0.5 = 285 mM) or ATP (S_0.5 = 11 mM) concentration was varied, suggesting cooperative binding of the two substrates.     

Interaction of trialkyltin(IV) chlorides with sarcoplasmic reticulum calcium ATPase

The interaction of the organotin compounds trimethyltin(IV) and tributyltin(IV) chlorides with the calcium pump from sarcoplasmic reticulum membranes was studied. It was found that the presence of calcium fully protects against the inhibitory effect of both organotin compounds. However, the apparent affinity of the protein for tributyltin chloride is two orders of magnitude higher than for trimethyltin chloride (K_0.5 values of 14 µ m and 1.4 m m , respectively). Studies of intrinsic fluorescence of the Ca²⁺‐ATPase and enzyme phosphorylation by ATP and Pi support the hypothesis that the inhibitory properties of trialkyltin compounds are due to the inhibition of calcium binding to the high‐affinity binding sites of the Ca²⁺‐ATPase. This suggests that there is a specific interaction between the trialkyltin compounds and the calcium binding sites of the protein. The effect of trialkyltin compounds on Ca²⁺‐ATPase was also addressed by differential scanning calorimetry to assess the thermal transition of the protein denaturation, and by infrared spectroscopy in the absorption region corresponding to the amide I band (1600–1700 cm^?1) to observe changes in the secondary structure of the protein. We conclude that the interaction of trialkyltin compounds with Ca²⁺‐ATPase reduces the affinity and cooperativity for calcium binding and, consequently, the inhibition of ATPase activity. These events are accompanied by changes in the secondary structure of the protein, including loss of α‐helix structure and a concomitant increase in protein aggregation or unfolding. The activity of trialkyltin compounds on the Ca²⁺‐ATPase is discussed in relation to their solubility in water and in the lipid phase. Copyright © 2012 John Wiley & Sons, Ltd.     

甲烷生物催化氧化制甲醇——几种化学物质对Methylosinus trichosporium 3011甲醇累积的影响

本文报导了在 Methylosinus trichosporium 3011细胞反应液中加入一些化学物质对甲醇累积的影响。结果表明,Na~+抑制甲烷单加氧酶的活性。当 M.trichosporium 3011细胞反应液中含高浓度磷酸根时,只有很少量甲醇累积下来。EDTA 和 Na_2EDTA 甲醇累积的促进作用小于甲酸钠或甲酸的作用。但由于 EDTA 不仅可以抑制甲醇脱氢酶的活性,而且可以螯合 Na~+,而甲酸则可使 NADH 辅酶再生。因此,反应液中含有2mM EDTA 和20mM 甲酸时甲醇累积量最高。     

Physical and enzymatic properties of nucleotide-depleted beef heart mitochondrial adenosine triphosphatase.

Tightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol-induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide-depleted enzyme restores oxidative phosphorylation in F1-deficient submitochondrial particles. Reconstitution of nucleotide-depleted F1 with the ATP analog (adenylyl-imidodiphosphate (AMP-PNP), almost 5 moles of AMP-PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi-ATP exchange reaction in F1-deficient submitochondrial particles. Incubation of the analog-labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time-dependent recovery of ATPase activity.     

Characteristics of the H(+)-translocating adenosine triphosphatase of Vibrio parahaemolyticus

We have characterized H(+)-translocating adenosine triphosphatase (ATPase) in membrane vesicles of Vibrio parahaemolyticus. The ATPase required high concentrations (about 0.5 M) of Na2SO4 (or other salts) for its maximum activity. Magnesium ion stimulated the ATPase activity, but Ca2+ did not. The activity of ATPase was inhibited by tetrachlorosalicylanilide, an H+ conductor, but not by another H+ conductor, carbonylcyanide-m-chlorophenylhydrazone. The activity was strongly inhibited by dicyclohexylcarbodiimide or Zn2+, and partially inhibited by azide, but not at all by vanadate.     

Frequency dependence of the shear moduli of spectrin studied using a multiple lumped resonator viscoelastometer

The frequency dependence (119-7860 Hz) of the storage and loss shear moduli, G' and G', of human erythrocyte spectrin dimer crude solutions at 22.5 degrees C has been measured using a Birnboim-Schrag multiple lumped resonator viscoelastometer. The measurements were carried out on solutions of ionic strength 1 mM containing 1.1-3.7 mg ml-1 spectrin. This corresponds to the terminal zone for G' and G'. Analysis of the data using the standard theory of hybrid relaxation spectra yields a relaxation time of 22.5 +/- 1 microseconds. The pure spectrin dimer relaxation time is estimated to be 16 +/- 3 microseconds. This result suggests that at an ionic strength of 1 mM, the spectrin dimers are extended and that the main relaxation process is simple end-over-end rotation.     

Active calcium treatment transport via coupling between the enzymatic and the ionophoric sites of Ca2+ + Mg2+-ATPase.

The 20K dalton fragment of Ca2+ + Mg2+-ATPase obtained from th tryptically digested sarcoplasmic reticulum has been further purified using Bio-Gel P-100. This removed low-molecular-weight UV-absorbing and positive Lowry-reacting contaminants. The ionophoric activity of the 20K fragment in both oxidized cholesterol and phosphatidylcholine:cholesterol membranes is unaltered by this further purification. The 20K selectivity sequence in phosphatidylcholine:cholesterol membrane is Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ Mg2+. Digestion of intact sarcoplasmic reticulum vesicles with trypsin, which results in the dissection of the hydrolytic site (30K) from the ionophoric site (20K), is shown to disrupt energy transduction between ATP hydrolysis and calcium transport. This further implicates the 20K dalton fragment as a calcium transport site. These data and previous evidence are discussed in terms of a proposed model for the ATPase molecular structure and the mechanisms of cation transport in sarcoplasmic reticulum.     

PROTOPORPHYRIN-SENSITIZED PHOTODYNAMIC MODIFICATION OF PROTEINS IN ISOLATED HUMAN RED BLOOD CELL MEMBRANES

Abstract— The photodynamic action of protoporphyrin on red cell ghosts is reflected by extensive cross-linking of membrane proteins to very high molecular weight protein aggregates. This process was studied with sepharose gel chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Most sensitive to this photodynamic effect are spectrin and band 2. 1, 2. 2, 2.3 and 4.1. polypeptides, which are cross-linked after very brief illumination periods, with a concomitant loss of spectrin-associated ATPase activity. Band 6 protein, representing the monomeric form of glyceraldehyde-3-phosphate dehydrogenase, is also very sensitive to protoporphyrin-induced cross-linking. The enzymatic activity decreased even faster than the amount of band 6 polypeptides, suggesting that modification(s) of the enzyme other than cross-linking, possibly by rapid photooxidation of a thiol group, may be responsible for inactivation.
Extracted and purified spectrin was cross-linked with about the same velocity as membrane-bound spectrin, reinforcing our previously drawn conclusion that membrane lipids are not involved in the cross-linking reaction. Eluted band 6 polypeptides on the other hand exhibited a relatively fast photo-oxidative modification but a much slower cross-linking to dimers and tetramers. This suggests that the membrane structure, e.g. the spectrin matrix may play an essential role in the incorporation of membrane-bound band 6 polypeptides in the high molecular weight cross-linked complex.     

Alkaline ribonuclease associated with polyribosomes in fibroblasts of experimental granulation tissue.

Alkaline ribonuclease (RNase) from polyribosomes derived from experimental granulation tissue has been purified 1900-fold through affinity chromatography. The preparation was homogeneous in sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis with an estimated molecular weight of 15 000. Purified RNase was completely inhibited in the presence of divalent ions Mg2+(100 mM) and Ca2+(100 mM) but activated slightly with Na+(50 mM). The enzyme is an endonuclease and the best substrates were poly(U), mixed RNA from yeast, rRNA from granulation tissue and poly(C). The estimated apparent Km-values were 0.037, 0.064, 0.13 and 0.27 g1-1, respectively. In polyribosomes RNase occurred in both free and p-chloromercuribenzoate (pCMB)-liberated forms. The total activity was at the highest but the proportion of the free activity minimal in the granulation tissue during the maximal synthesis of collagen.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate.

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.     

Quartz crystal microbalance with dissipation monitoring and surface plasmon resonance studies of carboxymethyl cellulose adsorption onto regenerated cellulose surfaces

Adsorption of anionic polyelectrolytes, sodium salts of carboxymethyl celluloses (CMCs) with different degrees of substitution (DS = 0.9 and 1.2), from aqueous electrolyte solutions onto regenerated cellulose surfaces was studied using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) experiments. The influence of both calcium chloride (CaCl(2)) and sodium chloride (NaCl) on CMC adsorption was examined. The QCM-D results demonstrated that CaCl(2) (divalent cation) caused significantly greater CMC adsorption onto regenerated cellulose surfaces than NaCl (monovalent cation) at the same ionic strength. The CMC layers adsorbed onto regenerated cellulose surfaces from CaCl(2) solutions exhibited greater stability upon exposure to flowing water than layers adsorbed from NaCl solutions. Both QCM-D and SPR results showed that CMC adsorption onto regenerated cellulose surfaces from CaCl(2) solutions increased with increasing CaCl(2) concentration up to the solubility limit (10 mM). Voigt-based viscoelastic modeling of the QCM-D data indicated that the CMC layers adsorbed onto regenerated cellulose surfaces had shear viscosities of η(f) ≈ 10(-3) N·s·m(-2) and elastic shear moduli of μ(f) ≈ 10(5) N·m(-2). Furthermore, the combination of SPR spectroscopy and QCM-D showed that the CMC layers contained 90-95% water. Adsorption isotherms for CMCs in CaCl(2) solutions were also obtained from QCM-D and were fit by Freundlich isotherms. This study demonstrated that CMC adsorption from CaCl(2) solutions is useful for the modification of cellulose surfaces.