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Biomedical subjects

M Cohn

Publications and source records attributed to M Cohn.

At least 181 records · Page 10Linked to original sources
PubMed1978

Metal dependence of the phosphate (oxygen)-water exchange reaction of Escherichia coli alkaline phosphatase. Kinetics followed by 31P(18O) NMR.

Phosphate-water oxygen exchange catalyzed by Escherichia coli alkaline phosphatase was monitored using the 18O shift on the 31P NMR signal of inorganic phosphate. Different kinetic patterns were observed with native zinc enzyme and with its cobalt analogue. For native enzyme at pH values ranging from 4.4 to 10.0, the distribution of 18O species in Pi, viz. P18O4, P18O316O,P18O216O2,P18O16O3,P16O4, with time is compatible with a kinetic scheme in which E-P, the noncovalent enzyme-phosphate complex, dissociates more rapidly than it forms the covalent complex E-P. For the cobalt enzyme at pH 6.8, the distribution of 18O species in Pi with time is different and leads to the conclusion that formation of E-P is more rapid than dissociation of Pi from E-P-A computer simulation gave good quantitative agreement with the observed distribution for the time course of the cobalt enzyme reaction when the ratio of the rate of formation of E-P to dissection of E-P was assumed to be 3 +/- 0.5.

Alkaline Phosphatase↗
PubMed1978

31P nuclear magnetic resonance spectra of the thiophosphate analogues of adenine nucleotides; effects of pH and Mg2+ binding.

The 31P nuclear magnetic resonance (NMR) spectra of the adenine nucleotide thio analogues, AMPS, ADPalphaS, ADPbetaS, ATPalphaS, ATPbetaS, and ATPgammaS, have been studied. Of primary interest were the increased sensitivity of chemical shifts to protonation and to magnesium binding of these analogues compared with the corresponding effects on AMP, ADP, and ATP. The usefulness of the characteristic NMR parameters of the thio analogues as probes in enzymatic reactions is discussed. The A2 diastereoisomers of ADPalphaS and ATPalphaS and the A and B isomers of ATPbetaS were enzymatically synthesized and the diasterioisomers of ADPalphaS and ATPbetaS were distinguished by their 31P NMR parameters. The stereospecificity of the enzymatic reactions involving the thio analogues of nucleotides can therefore be determined by 31P NMR. The difficulty involved in assigning phosphate ligands of Mg in MgADP and MgATP and their analogues on the basis of the magnitude of chemical shift changes (deltadelta) induced by Mg binding upon each 31P is discussed in the context of the anomalies in deltadelta of each 31P observed upon protonation of the terminal phosphate group. It is concluded that chemical shift data cannot yield unequivocal information concerning the absolute structure of metal complexes of nucleotides but can be used to monitor changes in metal chelation, for example, upon binding to enzyme.

Adenosine Triphosphate↗
PubMed1978

Guanosine 3',5'-monophosphate: a central nervous system regulator of analgesia.

The dibutyryl derivative of guanosine 3',5'-monophosphate (cyclic GMP), administered centrally, totally abolishes response to noxious stimuli without depressing the central nervous system. Analgesic properties of the nucleotide are not reversed by naloxone. Microinjected intracerebrally into different sites, dibutyryl cyclic GMP does not mimic the action of morphine. Pharmacological effects of dibutyryl cyclic GMP suggest that endogenous cyclic GMP modulates an inhibitory pain pathway distinct from that on which morphine acts.

Analgesia↗
PubMed1978

Isotopic (18O) shift in 31P nuclear magnetic resonance applied to a study of enzyme-catalyzed phosphate--phosphate exchange and phosphate (oxygen)--water exchange reactions.

An isotopic shift of the (31)P nuclear magnetic resonance due to (18)O bonded to phosphorus of 0.0206 ppm has been observed in inorganic orthophosphate and adenine nucleotides. Thus, the separation between the resonances of (31)P(18)O(4) and (31)P(16)O(4) at 145.7 MHz is 12 Hz and, in a randomized sample containing approximately 50% (18)O, all five (16)O-(18)O species are resolved and separated from each other by 3 Hz. Not only does this yield the (18)O/(16)O ratio of the phosphate but, more important, the (18)O-labeled phosphate in effect can serve as a double label in following phosphate reactions, for oxygen in all cases and for phosphorus, provided the oxygen does not exchange with solvent water. Thus, it becomes possible to follow labeled phosphorus or labeled oxygen continuously as reactions proceed. Rate studies involving (i) phosphorus and (ii) oxygen are illustrated by continuous monitoring of the exchange reactions between (i) the beta phosphate of ADP and inorganic phosphate catalyzed by polynucleotide phosphorylase and (ii) inorganic orthophosphate and water catalyzed by yeast inorganic pyrophosphatase. In the ADP-P(i) exchange, the P(i) ((18)O(4)) yielded an alpha P((16)O(3) (18)O) and a beta P((18)O(4)), proving that bond cleavage occurs between the alpha P and the alpha-beta bridge oxygen. Among the many additional potential uses of this labeling technique and its spectroscopic observation are: (i) different labeling of each phosphate group of ATP, (ii) to follow rate of transfer of (18)O from a nonphosphate compound such as a carboxylic acid to a phosphate compound, and (iii) to follow the rate of scrambling (for example, of the beta-gamma bridge oxygen of ATP to nonbridge beta P positions) and simultaneously the rate of exchange of the gamma P nonbridge oxygens with solvent water in various ATPase reactions.

Adenosine Diphosphate↗
PubMed1977

The derivation and characterization of neuronal cell lines from rat and mouse brain.

This study shows that permanent cell lines can be established from rat and mouse brain by direct tissue culture methodology without the aid of exogenous chemical or viral transforming agents. These cells were derived from specific areas of the brain, such as the cerebellum and hippocampus, at chosen times during fetal and neonatal development. Success in establishing neuronal cell lines was dependent upon the use of selection pressures designed to keep the background of glial cells and fibroblasts at a minimum. These manipulations included care in the choice and processing of the original tissue, utilization of cytotoxic anti-glial sera, and continuous manual isolation of cells with neuronal morphology. Slow-growing nerve cells were thus allowed to adapt spontaneously to culture with a minimum of competition from faster-adapting cell types. Many of these cell lines are judged to be neuronal on the basis of their electrical excitability and their characteristic surface antigens. The cells respond positively in a sodium flux assay which has been shown to correlate well with the ability to generate an action potential, and also express one or more of three antigens previously found to be specific for nerve cells.

Animals↗
PubMed1977

19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

The 19F NMR spectrum of Escherichia coli tRNA1Val in which [5-19F]uridine replaces 93% of all uridine and uridine-derived residues has been examined at 93.6 and 235 MHz. The resolution of 11 peaks and visibility of two additional shoulders at either frequency for the 14 FUra residues in the molecule attests to the excellence of 19F as a probe for the structure of tRNA1Val in solution. No significant gain in resolution was attained at the higher frequency. A comparison of the relative areas in the different regions of the 19F spectrum of mixed [FUra]tRNAs with that of [FUra]tRNA1Val suggests that the three single resonances at lowest field in the region 86.5 to 88.5 ppm upfield from trifluoroacetate correspond to the three invariant bases which form tertiary hydrogen bonds in all tRNAs, namely, 8 (U or s4U), 54 (T), and 55 (phi) in unsubstituted tRNAs.

Escherichia coli↗
PubMed1977

Magnetic resonance studies of the manganese guanosine di- and triphosphate complexes with elongation factor Tu.

Analysis of titration data of EF-Tu-GDP with Mn(II) where free and bound Mn(II) were determined by proton relaxation rate of water (PRR) yields one tight Mn(II) binding site and a value of 2 muM for the dissociation constant of Mn(II) from the EF-Tu-MnGDP complex, K'A. The dissociation constant of manganese nucleotide from the ternary EF-Tu-MnGDP complex, K2, 0.2 muM, was derived from the known value of Ks, the dissociation constant for the binary EF-Tu-GDP complex, and the titration data of the ternary complex with excess GDP as titrant. The apparent number, n, of rapidly exchanging water ligands coordinated to bound Mn(II) in the ternary complex EF-Tu-MnGDP is estimated from the frequency dependence of the PRR of the complex to be approximately 1. The value of n and the values of PRR enhancements, epsilont = 4.3 for EF-Tu-MnGDP at 21 degrees, 24.3 MHZ and epsilont = 4.1 for the ternary GTP complex, are unusually low for protein-Mn-nucleotide complexes. The antibiotic X5108 which induces GTPase activity in EF-Tu-MgGTP was shown to bind stoichiometrically to EF-Tu-MnGDP and thereby change the PRR enhancement of the complex from 4.3 to 7.4. The characteristic broad lines in the EPR spectra of Mn(II) nucleotides are strikingly narrowed upon binding of Mn(II) nucleotides to EF-Tu. The long electron spin relaxation times inferred from the EPR spectra indicate a limited access of solvent water to the first coordination sphere of Mn(II) in its EF-Tu-nucleotide complexes. The frequency dependence of the PRR indicates that the electron spin relaxation time, T1e, is the dominant process modulating the Mn(II)-H2O interaction of the EF-Tu-MnGDP complex and consequently determines the correlation time. The value of T1e, estimated from the PRR experiments to be 2.5 ns at 21 degrees, is consistent with the lower limit of T1e obtained from the line widths of the EPR spectrum of the complex. Upon binding of a stoichiometric quantity of the antibiotic X5108, the EPR spectrum of EF-Tu-MnGDP is severely broadened indicating greater access of solvent water to the manganese coordination sphere, i.e. an opening of the nucleotide binding site as already suggested by the increased PRR enhancement.

Binding Sites↗
PubMed1977

Asymmetric binding of the inhibitor di(adenosine-5') pentaphosphate (Ap5A) to adenylate kinase.

The effect of binding diadenosine pentaphosphate (Ap(5)A) to adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) has been investigated by (31)P nuclear magnetic resonance. The symmetric molecule, Ap(5)A, is a potent inhibitor of the adenylate kinase reaction, 2 ADP right arrow over left arrow ATP + AMP. Free Ap(5)A has two groups of signals in its (31)P nuclear magnetic resonance spectrum centered at 11.1 and 22.8 parts/million (ppm) upfield from 85% H(3)PO(4) that are assigned to the end (1-P and 5-P) and middle (2-, 3-, and 4-P) phosphates, respectively. Addition of Mg(2+) shifts the centers of these resonances to 11.7 and 22.3 ppm. The spectrum of Ap(5)A bound to porcine adenylate kinase shows five groups of signals centered at 10.9, 11.9, 20.5, 22.7, and 24.0 ppm; the resonances at 11.1 ppm (1-P and 5-P) and at 22.8 ppm (2-P and 4-P) are now clearly split, indicating asymmetric binding of Ap(5)A to the enzyme. The asymmetry is strikingly enhanced in enzyme-bound MgAp(5)A, which has resonances at 10.5, 12.5, 18.6, 22.7, and 25.6 ppm. By the addition of Mn(2+) to the enzyme.MgAp(5)A complex, the observed signals in increasing order of shifts were tentatively assigned to 1-P, 5-P, 4-P, 3-P, and 2-P, where the 3-, 4-, and 5-P positions correspond to the ATP-binding site on the enzyme. The asymmetry introduced in the phosphate chain of enzyme.MgAp(5)A is indicated by the (31)P chemical shift of 7 ppm between 2- and 4-P, which is one of the largest thus far observed for phosphate substrates bound noncovalently to enzymes.

Adenine Nucleotides↗