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C M Grisham

Publications and source records attributed to C M Grisham.

At least 19 recordsLinked to original sources
PubMed2001

Structural consequences of divalent metal binding by the adenylyl cyclase toxin of Bordetella pertussis.

Adenylyl cyclase toxin of Bordetella pertussis has been shown by several investigators to require Ca(2+) for its actions on target cells, but little is known about the nature and specificity of divalent metal binding to this novel toxin. Calcium is the preferred divalent metal since toxic actions are markedly reduced in the presence of divalent species other than calcium. Mn(2+) EPR was used to quantitate and characterize divalent metal binding and revealed that the toxin contains approximately 40 divalent metal sites, consisting of at least one class of high-affinity sites that bind Mn(2+) with a K(D) of 0.05 to 0.35 microM and one or more classes of lower affinity sites. Water proton relaxation data indicate that approximately 30 of these sites are completely inaccessible to bulk solvent. Our observations, together with the sequence homology between adenylyl cyclase toxin and the alkaline protease of Pseudomonas aeruginosa, indicate that the formation of five beta-sheet helices within the repeat domain of the toxin upon binding Ca(2+) is required for cell intoxication.

Adenylate Cyclase Toxin↗
PubMed1998

Influence of lipid on the structure and phosphorylation of protein kinase C alpha substrate peptides.

The structure and phosphorylation of two protein kinase C (PKC) alpha substrate peptides were investigated in varying lipid systems using enzyme activity assays and circular dichroism (CD) spectroscopy. The alpha-peptide, which exhibits the typical PKC alpha substrate motif and is based on the pseudosubstrate region of PKCalpha, was phosphorylated to a similar extent in bovine brain phosphatidylserine vesicles or diheptanoylphosphatidylcholine (PC7) micelles (both with 5 mol % 1,2-dioleoyl-sn-glycerol), whereas neuromodulin (NM)-peptide, which does not exhibit this motif by virtue of its primary structure, was phosphorylated to a much lesser extent in the PC7 micellar system. CD spectra of the peptides indicated that NM-peptide underwent a dramatic structural change in the presence of dimyristoylphosphatidylserine (DMPS) vesicles, whereas spectra acquired in PC7 micelles were similar to those acquired in buffer alone. No significant structural change was observed in the alpha-peptide in the presence of either lipid. PKC activity assays conducted with a series of NM-peptides successively substituted with nitroxide spin labels at each residue position suggested that several residues distal to the phosphorylation site are necessary for substrate recognition. The effect of these substitutions is not consistent with the binding of the NM-peptide to PKC in an extended structure, but is consistent with the binding of this peptide in a helical conformation. Furthermore, the docking of a helical NM-peptide to the substrate binding site of PKC suggests that the interaction is energetically feasible. These results suggest that PKC may recognize some non-linear substrate motifs and that lipid binding may convert a protein into a better PKC substrate.

Amino Acid Sequence↗
PubMed1994

31P NMR investigation of energy metabolism in perifused MMQ cells.

The MMQ cell line is a unique prolactin-secreting rat pituitary cell line. MMQ cells entrapped in agarose gel threads are metabolically active, as determined by the uptake and phosphorylation of creatine and the maintenance of high energy phosphates for over 15 h. Forskolin activates the catalytic subunit of adenylyl cyclase and, in MMQ cells, elevates the level of cAMP and stimulates prolactin secretion. 31P NMR spectroscopy was used to investigate the energy metabolism of the MMQ cells during stimulation by forskolin. The ability to measure small changes in the energy status of these cells was enhanced by increasing the PCr levels in the cells. Administration of forskolin to the perifused MMQ cells resulted in acute, reversible, and dose-dependent changes in the 31P NMR spectra of the cells within 12 to 24 min of the beginning of forskolin exposure. Several lines of evidence indicate that the changes observed in the MMQ cells are the composite result of the interaction of forskolin with adenylyl cyclase and the plasma membrane glucose transporter. Also, preincubation of the MMQ cells with the dopamine agonist, bromocriptine, attenuates the forskolin-stimulated decrease in the PCr resonance by approximately 50%. This attenuation indicates that the forskolin-stimulated changes in energy metabolism are probably related to the prolactin secretion process.

Adenylyl Cyclases↗
PubMed1992

Activation and inhibition of protein kinase C isozymes alpha and beta by Gd3+.

Gd3+ was evaluated as a probe for Ca2+ sites on protein kinase C (PKC) by studying its ability to replace Ca2+ in activation of PKC isozymes II (beta) and III (alpha) in the lipid systems phosphatidylserine/1,2-dioleoyl-sn-glycerol (PS/DO) and diheptanoylphosphatidylcholine (PC7)/DO. PKC beta was stimulated by Ca2+ or Gd3+ in PS/DO whereas activity in PC7/DO was independent of these metals. Thus, it is suggested that Gd3+ replaces Ca2+ at a site involving metal-lipid interactions. High concentrations of Ca2+ or Gd3+ inhibited activity in both lipid systems. Analysis of the Gd3+ inhibition in the PC7/DO system suggests that it is due to formation of GdATP, which competes at the MgATP site. Activity of PKC alpha was dependent on low concentrations of Ca2+ in both lipid systems. The ability of Gd3+ to substitute for Ca2+ could not be evaluated in the PS system due to the inability to completely remove contaminating Ca2+ without chelating buffers. Successful reduction of contaminating Ca2+ was achieved in the PC7 system but Gd3+ failed to substitute for Ca2+ in activating PKC alpha and only caused inhibition. This is consistent with binding of Gd3+ to a Ca2+ site at or near the active site of the enzyme rather than to a site on the lipid. These results indicate that interactions between PKC and Gd3+ are complex, involving occupation of more than one class of sites. Conditions for separately evaluating the individual sites can be manipulated by selection of isozyme and lipid system.

Animals↗
PubMed1992

High-affinity Ca(2+)- and substrate-binding sites on protein kinase C alpha as determined by nuclear magnetic resonance spectroscopy.

Water proton nuclear magnetic resonance (NMR) relaxation rates were used to identify metal sites on protein kinase C (PKC) isozymes alpha and beta using paramagnetic Gd3+ as a probe. The paramagnetic effect of Gd3+ on water proton relaxation was enhanced with PKC isozymes alpha and beta in the presence of diheptanoylphosphatidylcholine/1,2-dioleoyl-sn-glycerol (PC7/DO). The data are consistent with a single class of metal-binding sites on PKC beta and two classes of sites on PKC alpha: a single high-affinity site with a KD for Gd3+ of 0.2 microM and a larger class of sites with a lower affinity for Gd3+. Titration with Ca2+ abolished the observed enhancement of water proton relaxation by the PKC alpha.Gd3+ complex, consistent with displacement of Gd3+ by Ca2+. Titrations of the PKC alpha.Gd3+ complex with Co(NH3)4ATP, a substitution-inert analogue of ATP, caused a substantial decrease in the observed water proton relaxation enhancement, consistent with formation of a ternary enzyme.metal.substrate complex with a KPKC alpha.Gd.[CoATP] of 30-100 nM. Titration of the metal enzyme complex with a model peptide substrate derived from the pseudosubstrate sequence of PKC alpha caused a similar decrease in enhancement at stoichiometric concentrations consistent with the formation of a PKC alpha.Gd3+.peptide complex with a KPKC alpha.Gd.[peptide] of less than or equal to 13 nM. Titrations of the fully formed PKC alpha.Gd3+.peptide complex with Co(NH3)4ATP caused a further decrease in enhancement consistent with formation of a quaternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.

Adenosine Triphosphate↗
PubMed1992

Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR.

The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, these rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.

Animals↗
PubMed1991

Antibodies against the 53 kDa glycoprotein inhibit the rotational dynamics of both the 53 kDa glycoprotein and the Ca(2+)-ATPase in the sarcoplasmic reticulum membrane.

The purpose of this study is to better define the relationship of the 53 kDa glycoprotein (GP-53) of the sarcoplasmic reticulum (SR) to other SR proteins. Towards that end the effects of antibodies against GP-53 on the rotational dynamics of maleimide spin-labeled proteins of SR of rabbit skeletal muscle were investigated. The labeling protocol used in this study provided 1.6 +/- 0.3 moles spin label incorporated per 10(5) g SR protein. Labeling specificity studies indicated that nearly 70% of the label bound specifically to the Ca(2+)-ATPase, with the remainder bound to GP-53. Using saturation-transfer electron paramagnetic resonance (ST-EPR), it was determined that the rotational mobility (i.e., the rate of rotation) of the spin-labeled SR proteins decreased greater than 5-fold upon preincubation of MSL-SR with an antiserum against the GP-53, while preincubation of MSL-SR with preimmune serum had no effect. Preincubation of MSL-SR with a monoclonal antibody against the GP-53 produced a 4-fold decrease in the rotational mobility of the MSL-SR proteins compared to control measurements. Further, these effects showed a marked calcium dependence: the decrease in the rotational mobility of the MSL-SR proteins preincubated with anti-GP-53 antibodies in 500 microM Ca2+ was 3-6-fold greater than that of MSL-SR preincubated with antibodies in 5 mM EGTA. While MSL was bound to both Ca(2+)-ATPase and GP-53, model calculations indicated that the decreases observed in the rotational mobility of the MSL-SR proteins caused by the anti-GP-53 monoclonal antibodies were too large to be accounted for by effects on GP-53 alone. The calculations suggest that the rotational rate of Ca(2+)-ATPase was also diminished by anti-GP-53 monoclonal antibodies, indicating an interaction between GP-53 and Ca(2+)-ATPase in the SR membrane.

Antibodies, Monoclonal↗
PubMed1990

1H and 31P nuclear magnetic resonance and kinetic studies of the active site structure of chloroplast CF1 ATP synthase.

The interaction of nucleotides and nucleotide analogues and their metal complexes with Mn2+ bound to both the latent and dithiothreitol-activated CF1 ATP synthase has been examined by means of steady-state kinetics, water proton relaxation rate (PRR) measurements, and 1H and 31P nuclear relaxation measurements. Titration of both the latent and activated Mn(2+)-CF1 complexes with ATP, ADP, Pi, Co(NH3)4ATP, Co(NH3)4ADP, and Co(NH3)4AMPPCP leads to increases in the water relaxation enhancement, consistent with enhanced metal binding and a high ternary complex enhancement. Steady-state kinetic studies are consistent with competitive inhibition of CF1 by Co(NH3)4AMPPCP with respect to CaATP. The data are consistent with a Ki for Co(NH3)4AMPPCP of 650 microM, in good agreement with a previous Ki of 724 microM for Cr(H2O)4ATP [Frasch, W., & Selman, B. (1982) Biochemistry 21, 3636-3643], and a best fit KD of 209 microM from the water PRR measurements. 1H and 31P nuclear relaxation measurements in solutions of CF1 and Co(NH3)4AMPPCP were used to determine the conformation of the bound substrate analogue and the arrangement with respect to this structure of high- and low-affinity sites for Mn2+. The bound nucleotide analogue adopts a bent conformation, with the low-affinity Mn2+ site situated between the adenine and triphosphate moieties and the high-affinity metal site located on the far side of the triphosphate chain. The low-affinity metal forms a distorted inner-sphere complex with the beta-P and gamma-P of the substrate. The distances from Mn2+ to the triphosphate chain are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules or residues from the protein.

Adenosine Triphosphate↗
PubMed1990

Saturation transfer EPR measurements of the rotational motion of a strongly immobilized ouabain spin label on renal Na,K-ATPase.

The rotational motion of an ouabain spin label with sheep kidney Na,K-ATPase has been measured by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) measurements. Spin-labelled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per ATPase alpha beta dimer. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance which is characteristic of a strongly immobilized spin label. ST-EPR measurements of the spin labelled ATPase preparations yield effective correlation times for the bound labels of 209 +/- 11 microseconds at 0 degree C and 44 +/- 4 microseconds at 20 degrees C. These rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements with glutaraldehyde-crosslinked preparations indicated that the observed rotational correlation times predominantly represented the motion of entire Na,K-ATPase-containing membrane fragments, rather than the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The strong immobilization of the ouabain spin label will make it an effective paramagnetic probe of the extracellular surface of the Na,K-ATPase for a variety of NMR and EPR investigations.

Animals↗
PubMed1989

Nuclear Overhauser effect studies of the conformation of Co(NH3)4ATP bound to kidney Na,K-ATPase.

Transferred nuclear Overhauser effect measurements (in the two-dimensional mode) have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase [Klevickis, C., & Grisham, C.M. (1982) Biochemistry 21, 6979. Gantzer, M.L., et al. (1982) Biochemistry 21, 4083]. Nine unique proton-proton distances on ATPase-bound Co(NH3)4ATP were determined from the initial build-up rates of the cross-peaks of the 2D-TRNOE data sets. These distances, taken together with previous 31P and 1H relaxation measurements with paramagnetic probes, are consistent with a single nucleotide conformation at the active site. The bound Co(NH3)4ATP) adopts an anti conformation, with a glycosidic torsion angle of 35 degrees, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. Mn2+ bound to a single, high-affinity site on the ATPase lies above and in the plane of the adenine ring. The distances from enzyme-bound Mn2+ to N6 and N7 are too large for first coordination sphere complexes, but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

Magnetic resonance and kinetic studies of the partial complex and Component I subunit forms of Salmonella typhimurium anthranilate synthase.

Metal ion interactions of the monofunctional partial complex of Salmonella typhimurium anthranilate synthase were investigated using kinetic, NMR, and EPR methods. Mn2+ activates AS-partial complex in place of Mg2+, with a Km of 0.08 microM for Mn2+ and of 3.5 microM for Mg2+ in glutamine-dependent anthranilate synthase activity. The kinetics indicated that the metal interacts at the active site with chorismate, not glutamine. EPR and NMR water proton relaxation rate (PRR) studies supported this conclusion. EPR binding analysis showed that chorismate dramatically tightens Mn2+ binding by the partial complex. PRR experiments indicated that stoichiometric amounts of chorismate cause a substantial decrease in the enhancement of water relaxation by Mn2+, while millimolar amounts of glutamine have no effect. Analysis of the frequency dependence of water proton relaxation rates yielded dipolar correlation times of 2.5 x 10(-9) s and 4.1 x 10(-9) s for the Mn2+-partial complex and Mn2+-partial complex-chorismate complexes, respectively. These studies also indicated that chorismate binding reduces the number of fast-exchanging water molecules on enzyme-bound Mn2+ from 1 to 0.25. PRR experiments with the native bifunctional anthranilate synthase-phosphoribosyltransferase enzyme indicated the existence of additional Mn2+-binding sites which presumably function to activate the phosphoribosyltransferase activity of the Component II subunit.

Anthranilate Synthase↗
PubMed1988

NMR studies identify four intermediate states of ATPase and the ion transport cycle of sarcoplasmic reticulum Ca2+-ATPase.

Water proton nuclear relaxation measurements are used to detect and characterize four distinct intermediate states for Gd3+ bound to Ca2+ sites of sarcoplasmic reticulum Ca2+-ATPase in complexes with ATP analogues. In the absence of nucleotides, Gd3+ binds to two occluded Ca2+ transport sites on Ca2+-ATPase which have a low accessibility to solvent water. In the presence of the nonhydrolyzable ATP analogue, Co(NH3)4AMPPCP, a new state for bound Gd3+ (still occluded and with fewer waters of hydration) is observed. In the presence of Co(NH3)4ATP or ATP, two additional states for bound Gd3+ are detected in the NMR studies. The first of these probably represents an intermediate state for bound Gd3+ during ATP hydrolysis. The latter is the most occluded Gd3+ site yet observed in these studies and is probably analogous to the highly occluded E1-P state observed with CrATP [(1987) Biochim. Biophys. Acta 898, 313-322].

Adenosine Triphosphate↗
PubMed1988

NMR relaxation measurements detect four intermediate states of ATPase and transport cycle of sarcoplasmic reticulum Ca2+-ATPase.

At least four of the intermediate states of Ca2+-ATPase (and presumably ion transport) can be trapped and characterized using water proton relaxation measurements. Gd3+ binds to two occluded Ca2+ transport sites on Ca2+-ATPase which have a low accessibility to solvent water. In the presence of the MgATP analogue Co(NH3)4AMPPCP, a new state for bound Gd3+ with one less water of hydration) is observed. In the presence of Co(NH3)4ATP or ATP, two additional states for bound Gd3+ are detected by NMR, the first of which probably represents an intermediate state of ATP hydrolysis. The latter is the most occluded Gd3+ site yet observed in these studies and corresponds to the highly occluded E1-P state observed with CrATP (Vilsen and Andersen, Biochim. Biophys. Acta 898, 313 (1987).

Adenosine Triphosphate↗
PubMed1988

1H nuclear magnetic resonance studies of the conformation of an ATP analogue at the active site of Na,K-ATPase from kidney medulla.

1H nuclear magnetic relaxation measurements have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase [Klevickis, C., & Grisham, C. M. (1982) Biochemistry 21, 6979; Gantzer, M. L., Klevickis, C., & Grisham, C. M. (1982) Biochemistry 21, 4083] and that Mn2+ bound to a single, high-affinity site on the ATPase can be an effective paramagnetic probe for nuclear relaxation studies of the Na,K-ATPase [O'Connor, S. E., & Grisham, C. M. (1979) Biochemistry 18, 2315]. From the paramagnetic effect of Mn2+ bound to the ATPase on the longitudinal relaxation rates of the protons of Co(NH3)4ATP at the substrate site (at 300 and 361 MHz), Mn-H distances to seven protons on the bound nucleotide were determined. Taken together with previous 31P nuclear relaxation data, these measurements are consistent with a single nucleotide conformation at the active site. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. The glycosidic torsion angle is 35 degrees, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The bound Mn2+ lies above and in the plane of the adenine ring. The distances from Mn2+ to N6 and N7 are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗