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G M Hathaway

Publications and source records attributed to G M Hathaway.

At least 19 recordsLinked to original sources
PubMed2000

Quasi-linear gradients for capillary liquid chromatography with mass and tandem mass spectrometry.

Gradient elution, capillary liquid chromatography mass spectrometry was performed with linear, static gradients constructed by laminar flowing ten, 1.5 microL volume steps of decreasing organic concentration into tubing of small internal diameter. Sample loading, gradient formation, and sample elution were accomplished entirely by means of a commercially available micro-autosampler and single-syringe drive pump. The procedure was simple, fast, stable, and reproducible. Essentially linear gradients were produced without the use of additional valves, mixers, pumps or software. It took less than 10 minutes to form a gradient and less than 30 minutes to construct the set of individual buffer vials. The gradients were shown to be stable to storage. One hour after forming, peak retention times were reproduced to +/-0.5%. Long-term retention time reproducibility was found to vary by +/-2%. Chromatographic resolution was comparable or superior to that obtained by gradient elution with conventional dynamic mixing and split flow. The procedure was adapted with a 'peak parking' method which extended the time for generating peptide fragmentation data up to 10 minutes per peptide with the triple quadruple mass spectrometer. Using this technique, collision data were collected at the 25 femtomole level on nine of ten tryptic peptides in a single run.

Chromatography, Liquid↗
PubMed1996

Comparison of the properties of native and pentaammineruthenium(III)-modified xylanase.

Two xylanases, xynA of Bacillus pumilus and xyn II of Trichoderma reesei, were purified and then modified by the attachment of pentaammineruthenium, thereby resulting in the generation of a xylanase with veratryl alcohol oxidase activity. Hydrolytic activity of T. reesei xyn II on soluble xylans was unchanged by modification with pentaammineruthenium; however, modification of B. pumilus xynA greatly reduced xylan hydrolysis unless the active site of the xylanase was protected with xylose during the modification. The presence of histidine, cysteine, or reduced glutathione during xylan hydrolysis greatly increased the xylanase activity of the pentaammineruthenium-modified B. pumilus xylanase. Glycine, glutamic acid, methionine, or oxidized glutathione had no effect on xylanase activity.

Alcohol Oxidoreductases↗
PubMed1994

Characterization of modified and normal deoxyoligonucleotides by MALDI, time-of-flight mass spectrometry.

A commercially available, matrix-assisted, laser desorption/ionization (MALDI), time-of-flight mass spectrometer has been used for the successful characterization of picomole quantities of modified deoxyoligonucleotides. The procedure was found to be applicable to the analysis of a variety of modified synthetic structures that included oligos with an intact dimethoxytrityl-blocking group, with omega-aminohexyl- and omega-hexyl-modified bases and with alterations to the normal phosphate backbone: 2'-5' and phosphorothioate linkages. Use of an alpha-cyano-4-hydroxycinnamic acid/ammonium formate based matrix gave sharp peaks, sensitivity to 1 pmol, accuracy to 0.06% on average and avoided the necessity for cryogenic technique and/or specialized equipment. Mixtures of normal and modified oligonucleotides worked well without any special precautions as long as the modified oligonucleotides contained a polyanionic backbone. Modified oligonucleotides that contained an amide linkage in place of the usual phosphate ester link required a sinapinic acid- or esculetin-based matrix with no ammonium formate. Crude oligonucleotides could be rapidly analyzed as a quality-control measure using from 10 to 20 pmol of a typical 0.2-mumol scale synthesis.

Base Sequence↗
PubMed1992

Antagonism of synaptosomal calcium channels by subtypes of omega-agatoxins.

Venom of the funnel web spider Agelenopsis aperta inhibits the binding of 125I-omega-conotoxin GVIA (omega-CgTx) to calcium channels in chick brain synaptosomal membranes. Fractionation of the venom by liquid chromatography shows that this inhibitory activity is associated primarily with a diverse class of peptide toxins called omega-agatoxins (omega-Aga). Using binding inhibition as an assay, we purified and identified the novel, 76-amino acid toxin, omega-Aga-IIIA. Inhibition of 125I-omega-CgTx binding to chick synaptosomal membranes by omega-Aga-IIIA and omega-Aga-IIA is correlated with block of potassium-stimulated 45Ca entry into synaptosomes; omega-Aga-IA neither inhibits 125I-omega-CgTx binding nor 45Ca entry under identical conditions. omega-Aga-IIA and omega-Aga-IIIA are 20-30-fold more potent than omega-CgTx as antagonists of synaptosomal calcium channels. However, whereas omega-CgTx completely blocks 45Ca entry into synaptosomes at saturating concentrations, the omega-agatoxins maximally block only 60-70% of 45Ca entry. Pretreatment of synaptosomes with omega-Aga-IIIA occludes block of 45Ca entry by omega-CgTx. The results indicate that, while the omega-agatoxins bind to the entire population of omega-CgTx-sensitive calcium channels in chick synaptosomal membranes, they exert only a partial block of 45Ca flux. Such block could occur via two distinct mechanisms. Toxin binding may alter the kinetics of a homogeneous population of channels, resulting in lower overall conductance upon depolarization. Alternatively, the omega-agatoxins may bind to two distinct channel subtypes, only one of which is blocked as a result of toxin occupation.

Agatoxins↗
PubMed1991

State-of-the-art biomolecular core facilities: a comprehensive survey.

A survey of 124 protein and/or nucleic acid chemistry facilities has provided a basis for estimating the resources needed to establish a facility, the financial support needed to keep it operating, and the technical capabilities it might reasonably be expected to achieve. Based on these data, an average core facility occupied 870 ft2, was staffed by three full-time personnel, and was equipped with 4-5 major instrument systems. Because user fees generated an average of about $101,000/year in income compared with an average operating budget of about $197,000/year, even a facility that charged user fees would, on average, still require an annual subsidy of about $96,000. Although most government and industrial core facilities did not assess user fees, at least 83 of the 124 respondents did have a preestablished schedule of service charges that enabled a compilation to be made of the average cost of providing a number of typical facility analyses and syntheses. The greater than 100-fold range in charges assessed in core facilities for seemingly identical services was shown to result from the equally large range in the degree of subsidization of these laboratories. Although an average facility might be expected to offer four or five of the following six major services--amino acid sequencing, amino acid analysis, HPLC peptide isolation, peptide synthesis, fragmentation of proteins and DNA synthesis--less than 10% of the responding laboratories provided mass spectrometry, capillary zone electrophoresis, or RNA synthesis. With the exception of peptide synthesis, which had an average turn-around time of about 24 days, all other major services had turn-around times that averaged in the range of 4-9 days. Additional data are summarized regarding average sample throughput in core laboratories and the amount of protein that is needed for hydrolysis/amino acid analysis and sequencing.

Amino Acid Sequence↗
PubMed1984

Interaction of polyamines and magnesium with casein kinase II.

In reticulocytes, polyamines appear to be physiologically relevant activators of casein kinase II [Hathaway, G. M. and Traugh, J. A. (1984). J. Biol. Chem. 259, 7011-7015]. The mechanism by which polyamines and Mg2+ interact to activate casein kinase II has been investigated. These studies were conducted by holding ionic strength constant at 0.10 M. At low Mg2+ (2.5 mM), activation by spermine resulted in a 33% decrease in the apparent Km for casein. Under these conditions, a 2.3-fold increase in the maximum velocity of the reaction was observed, and half-maximal stimulation was obtained with 275 microM spermine. At a kinetically optimal Mg2+ concentration of 12.5 mM, the effects of spermine on Km and Vmax were reduced, and the concentration of spermine required to give 50% of maximal stimulation was increased to 750 microM. Kinetic data obtained at the two Mg2+ concentrations indicated that Mg2+ and spermine competed for the same form of the enzyme. Double-reciprocal plots of velocity versus Mg2+ concentration showed downward curvature at Mg2+ concentrations higher than 1 mM, and these results were interpreted as evidence for two binding sites on the enzyme with an apparent Km of 0.5 and 2.5 mM. Experiments carried out with ATP-Mg2+ in the absence of excess MgCl2 gave results consistent with an absolute requirement of the enzyme for the metal ion which could not be replaced by spermine. These results are consistent with the formation of an enzyme-activator complex. A model is proposed where spermine activates casein kinase II at one site on the enzyme at which MgCl2 can also bind, while a second, high-affinity site exists exclusively for the metal ion.

Adenosine Triphosphate↗
PubMed1984

Kinetics of activation of casein kinase II by polyamines and reversal of 2,3-bisphosphoglycerate inhibition.

The effects of polyamines on the catalytic activity of casein kinase II have been studied. Of the three polyamines tested (putrescine, spermidine, and spermine), spermine was the most effective at stimulating the enzyme. When physiological concentrations of potassium and magnesium were utilized, 50% activation was observed at 0.28 mM spermine or 0.70 mM spermidine. With mixtures of spermine and spermidine at physiological concentrations for the reticulocyte (0.04 and 1.06 mM, respectively), a 2.5-fold stimulation of casein kinase II activity was observed. In general, stimulation of the enzyme was dependent on salt concentration and it was necessary to hold ionic strength constant in order to separate specific activation by the polyamines from general salt activation. Optimum activation by polyamines was observed at low ionic strength and physiological concentrations of Mg2+. When beta-casein and eukaryotic initiation factors 2 or 3 were used as substrates, up to 3.5-fold stimulation of casein kinase II was observed. In the absence of polyamine, half-saturation of the enzyme by Mg2+ was observed at 1-3 mM MgCl2, a concentration much higher than required for ATP-Mg2+ complex formation. This dependence of the enzyme on Mg2+ was greatly diminished in the presence of spermine. Spermine decreased the apparent Km for casein and increased the maximum velocity of the reaction. Spermidine and spermine also effectively reversed inhibition by 2,3-bisphosphoglycerate. The significant activation by polyamines observed under conditions similar to those measured for the red cell suggest that the polyamines, spermidine and to a lesser extent spermine, function to regulate casein kinase II in vivo.

2,3-Diphosphoglycerate↗
PubMed1984

Regulation of casein kinase II by 2,3-bisphosphoglycerate in erythroid cells.

The hemoglobin regulator, 2,3-bisphosphoglycerate (glycerate-2,3-P2) has been shown to modulate the activity of casein kinase II from rabbit reticulocytes. Kinetic results were obtained with the exogenous substrate, beta-casein and the endogenous substrates, initiation factors (eIF-) 2 and 3. Experiments carried out to determine the interaction between glycerate-2,3-P2, Mg2+, substrate, and casein kinase II led to the following conclusions: 1) glycerate-2,3-P2 inhibition was competitive with respect to the protein substrate and noncompetitive with respect to ATP; 2) inhibition was not caused by depletion of ATP-Mg2+ as a consequence of Mg2+ complexation with glycerate-2,3-P2; 3) the response curve for glycerate-2,3-P2 was cooperative, but the cooperativity decreased as salt concentration increased; 4) glycerate-2,3-P2 inhibition was dependent on Mg2+ concentration up to about 5 mM MgCl2 but did not parallel glycerate-2,3-P2 X Mg2+ complex formation indicating that the Mg2+ dependence was not due to the formation of a glycerate-2,3-P2 X Mg2+ complex; 5) experiments with analogs of glycerate-2,3-P2 showed that the binary phosphate grouping was important in determining inhibition by glycerate-2,3-P2 while the presence of the carboxylate-phosphate pair was much less important; 6) low levels of glycerate-2,3-P2 stimulated phosphorylation of beta-casein, eIF-2, and eIF-3; the extent of stimulation was dependent on the affinity for casein kinase II and the level of the substrate. These effects were observed in the range of glycerate-2,3-P2 concentrations predicted for intracellular fluctuations in this metabolite. Therefore, it was concluded that glycerate-2,3-P2 could function both as an activator and an inhibitor of casein kinase II in the erythroid cell by binding at the substrate binding site.

2,3-Diphosphoglycerate↗
PubMed1983

Casein kinase I.

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Animals↗
PubMed1983

Casein kinase II.

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Adenosine Triphosphate↗
PubMed1981

Identification of the catalytic subunit of casein kinase II by affinity labeling with 5'-p-fluorosulfonylbenzoyl adenosine.

Casein kinase II, purified from reticulocytes, was covalently labeled with the ATP affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine. The reaction was monitored by the decrease in enzyme activity and showed saturation kinetics with respect to the sulfonyl compound. This suggested a rapid equilibrium was established between the enzyme and affinity reagent prior to a slower, rate-determining step in the overall inactivation process. The enzyme was protected from the covalent modification by ATP and a series of ATP analogs. Their effectiveness in preventing inactivation by the affinity labeling reagent paralleled their ability to function as inhibitors of the phosphotransferase reaction. When radioactive p-fluorosulfonyl [14C]benzoyl adenosine was used to inactivate the enzyme, the alpha subunit was labeled and incorporation of radioactivity into the alpha subunit was blocked when ADP was included in the reaction mixture. Thus the ATP binding site of casein kinase II was shown to be contained within the domain of the alpha subunit of the alpha 2 beta 2 complex.

Adenosine↗
PubMed1980

Inhibition of casein kinase II by heparin.

Casein kinase II, a cyclic nucleotide-independent protein kinase from rabbit reticulocytes, was shown to be inhibited by heparin. Heparin specifically inhibited the enzyme and had no effect on other protein kinases, including casein kinase I, the type I and II cAMP-dependent protein kinases, protease-activated kinase I, and the hemin-controlled repressor. Heparan sulfate was found to be 40-fold less effective than heparin towards casein kinase II; other acid mucopolysaccharides had little or no effect on the enzymatic activity. Steady state studies revealed that heparin acted as a competitive inhibitor with respect to the substrate, casein. A value of 20 ng/ml or about 1.4 nM was obtained for the apparent Ki. The inhibition was not reversed by ATP and varying the ATP and heparin concentrations in the assay only altered the maximum velocity.

Animals↗
PubMed1978

Oxidation of low-spin iron(II) porphyrins by molecular oxygen. An outer sphere mechanism.

Hexacoordinate low-spin iron(II) porphyrins are oxidized by molecular oxygen in amine solvents at room temperature by a process that is acid dependent. The visible and NMR spectra of solutions of the iron complexes and the influence of axial ligands upon the rate of oxidation are consistent with an outer sphere mechanism that entails the dissociation of a protonated 1:1 iron porphyrin-oxygen pi complex as the rate-limiting step.

Chemical Phenomena↗