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J A Adams

Publications and source records attributed to J A Adams.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effect of groundwater flow on remediation of dissolved-phase VOC contamination using air sparging.

This paper presents two-dimensional laboratory experiments performed to study how groundwater flow may affect the injected air zone of influence and remedial performance, and how injected air may alter subsurface groundwater flow and contaminant migration during in situ air sparging. Tests were performed by subjecting uniform sand profiles contaminated with dissolved-phase benzene to a hydraulic gradient and two different air flow rates. The results of the tests were compared to a test subjected to a similar air flow rate but a static groundwater condition. The test results revealed that the size and shape of the zone of influence were negligibly affected by groundwater flow, and as a result, similar rates of contaminant removal were realized within the zone of influence with and without groundwater flow. The air flow, however, reduced the hydraulic conductivity within the zone of influence, reducing groundwater flow and subsequent downgradient contaminant migration. The use of a higher air flow rate further reduced the hydraulic conductivity and decreased groundwater flow and contaminant migration. Overall, this study demonstrated that air sparging may be effectively implemented to intercept and treat a migrating contaminant plume.

Air↗
PubMed2000

Substrate specificity of the oncoprotein v-Fps: site-specific mutagenesis of the putative P+1 pocket.

Based on the X-ray structure of the insulin receptor kinase [Hubbard, S. R. (1997) EMBO J. 16, 5572-5581], Arg-1130 in the oncoprotein v-Fps, a nonreceptor tyrosine protein kinase, is predicted to interact with the P+1 glutamate in substrate peptides. To determine whether this residue is an important recognition element in v-Fps, Arg-1130 was substituted with leucine (R1130L) and glutamic acid (R1130E). The ability of these mutants to phosphorylate the peptide EAEIYXAIE, where X is glutamic acid, alanine, or lysine, was assessed. A comparison of the rates of peptide phosphorylation under limiting substrate concentrations (i.e., k(cat)/K(m) conditions) indicates that substrate specificity is altered by the electrostatic environment of the P+1 pocket. When the pocket displays a positive charge (Arg-1130; wild type), no charge (R1130L), or a negative charge (R1130E), v-Fps prefers to phosphorylate the glutamate peptide over the lysine peptide by a 200:1, 9:1, or 1:1 margin. While k(cat)/K(m) for the glutamate peptide is 50-fold higher for wild type compared to R1130E, k(cat)/K(m) for the lysine peptide is 3-fold higher for R1130E compared to wild type, a 150-fold change in relative substrate specificity. Analysis of the individual steps in the kinetic mechanism using viscosometric techniques indicates that the wild-type enzyme binds the glutamate peptide 3-fold better than the alanine peptide and, at least, 10-fold better than the lysine peptide. For R1130L, this margin range is reduced substantially, and for R1130E, no binding preference is observed. Nonetheless, the lysine peptide binds, at least, 4-fold better to R1130E than to wild type, and the glutamate peptide binds 3-fold poorer to R1130E than to wild type. The mutants lower the phosphoryl transfer rate by 4-30-fold for the three peptides, suggesting that Arg-1130 helps to position the tyrosine for optimum catalysis. The data indicate that a single mutation in v-Fps can alter significantly the relative substrate specificity by about 2 orders of magnitude with, at least, 50% of this effect occurring through relative changes in peptide binding affinity.

Amino Acid Sequence↗
PubMed2000

Megakaryopoiesis in vitro in myelodysplastic syndromes and acute myeloid leukaemia: effect of pegylated recombinant human megakaryocyte growth and development factor in combination with other growth factors.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG-rHuMGDF combined with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61-positive cells in suspension cultures increased with PEG-rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL-3 and/or SCF were added to PEG-rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU-Mk growth was poor overall, but could be enhanced by PEG-rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG-rHuMGDF can be augmented by IL-3 and SCF in many MDS and AML patients.

Adolescent↗
PubMed2000

Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives.

The binding of the methylanthraniloyl derivatives of ATP (mant-ATP), ADP (mant-ADP), 2'deoxyATP (mant-2'deoxyATP), and 3'deoxyATP (mant-3'deoxyATP) to the catalytic subunit of protein kinase A was studied to gain insights into the mechanism of nucleotide binding. The binding of the mant nucleotides leads to a large increase in fluorescence energy transfer at 440 nm, allowing direct measurements of nucleotide affinity. The dissociation constant of mant-ADP is identical to that for ADP, while that for mant-ATP is approximately threefold higher than that for ATP. The dissociation constant for mant-3'deoxyATP is approximately fivefold higher than that for 3'deoxyATP while derivatization of 2'deoxyATP does not affect affinity. The time-dependent binding of mant-ATP, mant-2'deoxyATP, and mant-ADP, measured using stopped-flow fluorescence spectroscopy, is best fit to three exponentials. The fast phase is ligand dependent, while the two slower phases are ligand independent. The slower phases are similar but not identical in rate, and have opposite fluorescence amplitudes. Both isomers of mant-ATP are equivalent substrates, as judged by reversed-phase chromatography, although the rate of phosphorylation is approximately 20-fold lower than the natural nucleotide. The kinetic data are consistent with a three-step binding mechanism in which initial association of the nucleotide derivatives produces a highly fluorescent complex. Either one or two conformational changes can occur after the formation of this binary species, but one of the isomerized forms must have low fluorescence compared to the initial binary complex. These data soundly attest to the structural plasticity within the kinase core that may be essential for catalysis. Overall, the mant nucleotides present a useful reporter system for gauging these conformational changes in light of the prevailing three-dimensional models for the enzyme.

Adenine Nucleotides↗
PubMed2000

Noninvasive motion ventilation (NIMV): a novel approach to ventilatory support.

A motion platform was developed that oscillates an animal in a foot-to-head direction (z-plane). The platform varies the frequency and intensity of acceleration, imparting periodic sinusoidal inertial forces (pG(z)) to the body. The aim of the study was to characterize ventilation produced by the noninvasive motion ventilator (NIMV) in animals with healthy and diseased lungs. Incremental increases in pG(z) (acceleration) with the frequency held constant (f = 4 Hz) produced almost linear increases in minute ventilation (VE). Frequencies of 2-4 Hz produced the greatest VE and tidal volume (VT) for any given acceleration between +/-0.2 and +/-0.8 G. Increasing the force due to acceleration produced proportional increases in both transpulmonary and transdiaphragmatic pressures. Increasing transpulmonary pressure by increasing pG(z) produced linear increases in VT, similar to spontaneous breathing. NIMV reversed deliberately induced hypoventilation and normalized the changes in arterial blood gases induced by meconium aspiration. In conclusion, a novel motion platform is described that imparts periodic sinusoidal acceleration forces at moderate frequencies (4 Hz) to the whole body in the z-plane. These forces, when properly adjusted, are capable of highly effective ventilation of normal and diseased lungs. Such noninvasive ventilation is accomplished at airway pressures equivalent to atmospheric or continuous positive airway pressure, with acceleration forces less than +/-1 G(z).

Abdomen↗
PubMed2000

Hemodynamic effects of periodic G(z) acceleration in meconium aspiration in pigs.

The hemodynamic effects of periodic acceleration (pG(z)), induced in the spinal axis with noninvasive motion ventilation (NIMV), were studied in a piglet model of pulmonary hypertension associated with meconium aspiration. Animals (n = 12) were anesthetized, paralyzed, intubated, and supported by conventional mechanical ventilation (CMV). Thirty minutes after tracheal instillation of meconium solution (6 ml/kg), either CMV (n = 6) was continued or NIMV (n = 6) was initiated. Changes in systemic and pulmonary hemodynamics and arterial blood gases were tracked for 2 h after aspiration. Thermodilution, cardiac output, and heart rate were not significantly different after meconium aspiration in the pG(z) group relative to the CMV controls. Aortic pressure and systemic vascular resistance were significantly lower (approximately 30%) after meconium aspiration in NIMV animals relative to CMV animals. Pulmonary arterial pressure and pulmonary vascular resistance were also significantly lower, by 100%, after aspiration of meconium in the NIMV animals compared with the CMV controls. Meconium aspiration significantly decreased total respiratory compliance by approximately 50% and increased total respiratory resistance by approximately 100% in both CMV and NIMV animals, but such alterations did not differ between the two groups. Both CMV and NIMV satisfactorily supported ventilation in these paralyzed animals. In conclusion, NIMV through pG(z) in the spinal axis decreased systemic and pulmonary vascular resistance in piglets after meconium aspiration.

Acceleration↗
PubMed1999

Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique.

The dissociation rate constants for the two products of the reaction catalyzed by protein kinase A, ADP and phosphopeptide, were measured using a catalytic trapping technique to determine the role of product release in enzyme turnover. The enzyme was preequilibrated with ADP, and the reaction was initiated with a peptide substrate, LRRASLG, and ATP in a rapid quench flow instrument. At high, free magnesium concentrations (>2 mM), the large 'burst' in phosphopeptide production disappears, and, at low concentrations of free magnesium (0.5-1 mM), the kinetic transients become sigmoidal prior to the linear turnover phase. Increasing the concentrations of ATP or ADP did not influence the shape of the kinetic transients in the first 20 ms. ADP preequilibration protects the enzyme from inhibition by the covalent inactivator p-fluorosulfonylbenzoyl 5'-adenosine at 0.5 mM free magnesium, indicating that a competent E. ADP complex forms at low metal concentrations and the sigmoidal behavior in the catalytic trapping experiment is not due to free enzyme at high ATP concentrations. Simulations of the data indicate that ADP release is rate-limiting for turnover at high magnesium concentrations, but, at lower physiological levels of 0.5 and 1 mM, the off rate of ADP is 3- and 2-fold higher than k(cat), respectively. In contrast, the initial portions of the kinetic transients at 0.5 mM free magnesium were unaffected by phosphopeptide preequilibration, indicating that the release rate of this product is significantly larger than turnover. The transient kinetic data, coupled with a previous report [Shaffer and Adams (1999) Biochemistry 38, 5572-5581], support a phosphorylation mechanism under physiological magnesium concentrations that incorporates two partially rate-determining conformational changes, one prior to and one after the phosphoryl transfer step. We propose that the initial step activates the enzyme through key positioning of one or more active-site residues and the second step relaxes this conformation, a prerequisite for a subsequent catalytic cycle.

Adenosine↗
PubMed1999

An ATP-linked structural change in protein kinase A precedes phosphoryl transfer under physiological magnesium concentrations.

The kinetic mechanism for the catalytic subunit of protein kinase A was evaluated using physiological concentrations of free magnesium (0.5 mM) and a rapid quench flow technique. When the enzyme is pre-equilibrated with ATP, the peptide substrate, LRRASLG (Kemptide), is phosphorylated in a biphasic manner with a rapid, exponential "burst" phase (kb) followed by a slower, linear phase (kL) that corresponds to the steady-state kinetic rate. Both the amplitude and the substrate-rate dependence of the initial, burst phase indicate that the rate of phosphoryl transfer is fast (approximately 500 s-1) and does not limit turnover (45 s-1). Viscosity studies indicate that, while Kemptide is in rapid equilibrium, ATP does not exchange rapidly with the active site and kcat/KATP is limited by the rate constant for nucleotide encounter. When the pre-steady-state kinetic experiments are initiated with ATP, a lag phase is observed at low ATP concentrations consistent with rate-limiting association. At high ATP concentrations (>1 mM), a burst phase is observed but the rate and amplitude are low on the basis of the bimolecular rate constant for ATP association and the rate constant for phosphoryl transfer. The kinetic data indicate that the phosphoryl transfer step is fast at physiological magnesium concentrations, but an ATP-linked conformational change precedes this step, limiting the burst phase rate constant. Simulations of the pre-steady-state kinetic transients indicate that turnover (45 s-1) is limited both by net product release (70 s-1) and by this structural change (170 s-1). This structural change may also occur at high free magnesium concentrations, but it must be significantly faster than 170 s-1 and, consequently, not rate-limiting for turnover (kcat = 20 s-1 at 10 mM free Mg2+). We propose that this conformational event is an obligatory component of the kinetic pathway and includes a movement of the catalytic residues necessary for supporting phosphoryl group donation.

Adenosine Triphosphate↗
PubMed1999

Proton beam therapy for age-related macular degeneration: development of a standard plan.

Age-related macular degeneration is the leading cause of blindness in developing countries. Irradiating the exudative form, in which a choroidal neovascular membrane develops in the subfoveal area, is presently a treatment under investigation. In 1995, Massachusetts General Hospital, collaborating with Massachusetts Eye and Ear Infirmary, initiated a protocol to treat SCNV membranes using the proton beam at the Harvard Cyclotron Laboratory and the EYEPLAN program with a light-field setup. EYEPLAN requires the axial eye length, membrane dimensions, and manipulation of the eye to include a 4.0-mm radial margin around the membrane so that the aperture margin (50% isodose line on the posterior retina) abuts the inferior aspect of the limbus. Review of 100 individually prepared plans showed that 95% of the fabricated apertures were circular (aspect ratio < 1.095) with diameters 9.5 to 15.0 mm. This information was used to develop an automated standard plan. Thirty-nine plans were developed for axial lengths ranging from 21.0 to 25.0 mm and membrane sizes from 1.5 to 6.75 mm in the usual way as the reviewed ones. Circular targets were outlined centered on the fovea. Distal and proximal 90% ranges (modulation) to the target, and doses to macula, optic disc, lens, ciliary body, retina, and globe were calculated. An automated standard plan requiring the same input data, but avoiding the need for individual plans, was developed. The program outputs the aperture diameter, fixation angle for the light-field setup, range and modulation, and calculates dose to the macula and optic nerve and percentage of retina receiving > or = 50% and > or = 90% of the prescribed dose. Individual plans require approximately 1.5 hours; the standard plan, 5 minutes. The standard plan could have treated 86% of the reviewed plans. The automated plan provides accurate and efficient treatment parameters for the majority of patients.

Fluorescein Angiography↗
PubMed1999

Past physical abuse is significantly correlated with pregnancy as an adolescent.

STUDY OBJECTIVE: To determine which type or types of childhood abuse are associated with adolescent pregnancy and other adverse health outcomes. DESIGN: 100 females between the ages of 12 and 24 years of age, attending either an adolescent/young adult clinic (for ages 12 to 24), or teen obstetrics clinic (for ages 13 to 18) at an urban academic medical center, were recruited to participate in an interview study during their clinic visit. The clinician or research assistant used a 186-item structured interview form to record answers to detailed questions on abuse, in addition to traditional questions about the patient's feelings on home, education, activities, drugs and depression, sex and suicide (HEADS). RESULTS: 32% of subjects reported a history of sexual abuse, 29% reported physical abuse, and 46% reported past emotional abuse. The frequency of any past abuse was significantly higher in girls who had ever been pregnant (29 of 50, 58%), compared with "never pregnant" girls (19 of 49, 38%)( P < .05). A past history of physical abuse (P = .04), but not sexual or emotional abuse, was significantly associated with pregnancy as a teen. Other factors significantly correlated with teen pregnancy were: frequent alcohol use, older age, and Mexican-American or African-American ethnicity. Past history of abuse was significantly correlated with depression, suicidal thoughts, absent father, school failure, alcohol and tobacco use, and delinquency. CONCLUSIONS: A history of past physical abuse is strongly associated with adolescent pregnancy, and questions about all types of abuse should be routinely asked of adolescent patients.

Adolescent↗
PubMed1999

Reduced bone mineral density in young adults following cure of acute lymphoblastic leukaemia in childhood.

Bone mineral density (BMD), serum osteocalcin and type I collagen C-telopeptide (ICTP) were assessed in a cohort of 31 (16 males) adults who had received cranial irradiation in childhood as part of their treatment for acute lymphoblastic leukaemia (ALL). Markers of bone turnover were compared with those of 35 age and body mass index (BMI) matched young adults (18 male). Growth hormone status had previously been determined using an insulin tolerance test and arginine stimulation test. Eight patients were classified as severe growth hormone deficiency (group 1), 12 patients as growth hormone insufficient (group 2) and 11 patients as normal (group 3). Vertebral trabecular BMD, lumbar spine and femoral neck integral BMD and forearm cortical bone mineral content (BMC) was measured 17.8 (6.8-28.6) years after cranial irradiation and was expressed as Z (standard deviation) scores. There was a significant reduction in vertebral trabecular BMD (median Z score -1.25, P < 0.001), in lumbar spine integral BMD (median Z score -0.74, P = 0.001), in forearm cortical BMC (median Z score -1.35, P < 0.001), and less so in femoral neck integral BMD (median Z score -0.43, P = 0.03). There was no difference among the growth hormone status groups for the following BMD measurements: vertebral trabecular BMD, lumbar spine integral BMD or femoral neck integral BMD (P = 0.8, P = 0.96 and P = 0.4 respectively). There was only a marginal significant difference for BMD at the wrist between growth hormone status groups (P = 0.04). There was no correlation between the BMD measurements with time since or age at diagnosis and no difference in markers of bone turnover between patients and controls; median serum osteocalcin 13.3 and 12.0 ng ml (P = 0.7), respectively, and for ICTP 5.0 and 4.9 microg L (P = 0.67) respectively. In conclusion, there is a highly significant reduction in BMD in young adults following treatment for ALL in childhood. The reduction in BMD affects both trabecular and cortical bone but did not seem to be related to time since diagnosis, age at diagnosis, or current growth hormone status. Possible explanations include a direct effect of chemotherapy, steroids or both on bone during childhood and hence an effect on the accretion of bone mass. In view of the risk of fractures in patients with osteopenia, adults treated for ALL in childhood may be at an increased risk of bone fractures later in life irrespective of the underlying cause of the osteopenia and thus intervention should be considered.

Adolescent↗
PubMed1999

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) on megakaryopoiesis in patients with aplastic anaemia.

Recombinant human megakaryocyte growth and development factor (rHuMGDF), a truncated form of the Mpl ligand, stimulates megakaryopoiesis both in vitro and in vivo. We describe the in vitro effect of pegylated recombinant human MGDF (PEGrHuMGDF) alone and in combination with other haemopoietic growth factors (G-CSF, GM-CSF, IL3, IL6, erythropoietin, SCF) on megakaryopoiesis in bone marrow from 11 normal subjects and 19 patients with aplastic anaemia (AA). We used semi-solid cultures to assess megakaryocyte colony growth (CFU-Mk) and 7 d suspension cultures to assess production of platelet glycoprotein IIIa (CD61) positive cells. CFU-Mk growth from normal marrow increased 3-4-fold and CD61+ve cells in suspension culture increased 8-10-fold with the addition of 10 ng/ml PEGrHuMGDF. In normal subjects growth factor combinations further increased responses in suspension culture, PEGrHuMGDF + SCF, PEGrHuMGDF + IL3 and PEGrHuMGDF + SCF + IL3 + Epo (P<0.05). IL6, GM-CSF, G-CSF or Epo added with PEGrHuMGDF did not consistently give this increase. CFU-M. growth from AA marrow remained very low in the presence of PEGrHuMGDF, with or without the addition of other growth factors. CD61+ve cells in suspension culture were, however, increased in the presence of PEGrHuMGDF alone in 12/19 AA cases. Of the 12 patients responsive to PEGrHuMGDF, nine were tested with additional growth factors and further responses were seen in six. In the AA cases PEGrHuMGDF+SCP and PEGrHuMGDF+SCF+IL3+Epo gave the highest responses. These data suggest that PEGrHuMGDF, alone or in combination with SCF and/or IL3, can enhance megakaryocyte proliferation in some patients with aplastic anaemia and may therefore have a role in the treatment of thrombocytopenia in these cases.

Adolescent↗
PubMed1999

Hyperleptinaemia in young adults following cranial irradiation in childhood: growth hormone deficiency or leptin insensitivity?

OBJECTIVE: In order to explore the mechanism of obesity in long-term survivors of childhood leukaemia, fat mass, lean body mass and serum leptin were assessed in a cohort of 32 (17 males) adults who had received cranial irradiation (XRT) in childhood as part of their treatment for acute lymphobiastic leukaemia (ALL), and compared with 35 age and body mass index (BMI) matched young adults (18 male). DESIGN: Thirty-one patients and 18 controls had fat mass and lean body mass assessed by dual x-ray absorptiometry (DEXA), using a lunar DPX-L scanner. Serum leptin concentrations were also measured in 27 patients and all controls. Growth hormone status had previously been determined using an insulin tolerance test and arginine stimulation test. Nine patients were classified as severe growth hormone (GH) deficient (group 1), 12 patients as GH insufficient (group 2) and 11 patients as normal (group 3). RESULTS: BMI and absolute fat mass were not significantly different between the patients and controls regardless of their gender (P = 0.1 and P = 0.14 respectively). In contrast, absolute lean mass was significantly reduced (P < 0.01) and leptin concentrations were significantly increased (P < 0.001) in patients compared with controls. BMI, fat mass and leptin concentrations but not lean mass were significantly different between the three GH status groups (P < 0.01, P < 0.01, P = 0.004, and P = 0.67 respectively). When leptin concentrations were expressed per unit of fat mass, they were increased in the patients compared with the controls (P = 0.03) with significant differences between the GH status groups (P = 0.004), being significantly higher in the severe GH deficient group. CONCLUSIONS: Young adults who receive cranial irradiation in childhood are prone to GH deficiency and hyperleptinaemia. The pathophysiological significance of the hyperleptinaemia remains to be established but it has occurred either as a consequence of radiation induced hypothalamic damage or GH deficiency.

Adolescent↗
PubMed1998

Mutations in the activation loop tyrosine of the oncoprotein v-Fps.

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.

Animals↗
PubMed1998

A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps.

The activity of the kinase domain of the oncoprotein v-Fps was found to be sensitive to the concentration of magnesium ions. Plots of initial velocity versus free magnesium concentration are hyperbolic and do not extrapolate to the origin at stoichiometric ATP-Mg, indicating that there are two sites for metal chelation on the enzyme and the second is nonessential for catalysis. The second metal is strongly activating and increases the reaction rate constant almost 20-fold from 0.5 to 8.3 s-1 using 0.2 mM ATP-Mg and 1 mM peptide, EAEIYEAIE. This increase in rate is due to a large increase in the apparent affinity of ATP-Mg at high magnesium concentrations. At 0.5 and 10 mM free Mg2+, KATP-Mg is 3.6 and 0.22 mM, respectively. Extrapolation of the observed affinity of ATP-Mg to zero and infinite free metal indicates that KATP-Mg is greater than 8 mM in the absence of the second metal and 0.1 mM in the presence of the second metal, a minimum 80-fold enhancement. By comparison, free levels of the divalent ion do not influence maximum turnover (kcat) and have only a 2-fold effect on the Km for the peptide substrate between 0.5 and 20 mM free Mg2+. Viscosometric studies indicate that free Mg2+ does not influence the rates of phosphoryl transfer or net product release above 0.5 mM but does affect directly the dissociation constant for ATP-Mg. The Kd for ATP-Mg in the absence and presence of the second metal ion is >32 and 0.4 mM, respectively. At high magnesium concentrations, ATP-Mg and the peptide substrate bind independently, while at lower concentrations (0.5 mM), there is significant negative binding synergism suggesting that the second metal may help to reduce charge repulsion between ATP-Mg and the peptide. The data indicate that the first metal is sufficient for phosphoryl transfer. While the second metal could have some influence on phosphoryl transfer or product binding, it is a potent activator that functions minimally by controlling ATP-Mg binding.

Adenosine Triphosphate↗
PubMed1998

A mathematical model for ligand/receptor/G-protein dynamics and actin polymerization in human neutrophils.

A mathematical model is proposed for describing the dynamics of the chemotactic peptide-stimulated actin polymerization response in human neutrophils. The response pathway utilizes the guanine nucleotide binding protein (G-protein) signal transduction cascade common to many receptor systems and allows adaptation in the continued presence of ligand. The development of such a model is an important first step toward understanding, predicting, and ultimately manipulating neutrophil responses. The model is divided into two parts, ligand/receptor/G-protein dynamics and the actin polymerization mechanism. Fast (receptor precoupled to G-protein) and slow (free receptor) signaling pathways involving ligand/receptor/G-protein interactions produce an activated signaling molecule. The actin polymerization mechanisms utilizes an actin binding protein which complexes with actin monomer and inhibits polymerization in an unstimulated cell. During stimulation, the activated signaling molecule enhances the dissociation of monomer/binding protein complexes, allowing the actin polymerization response to occur. The fast and slow signaling pathways are predicted to have different roles in controlling the time course of this actin polymerization. Additionally, precoupled receptors are predicted to have a larger ligand association rate constant than non-precoupled (free) receptors. Model simulations agree with many of the experimentally observed characteristics of both the stimulated F-actin response and ligand/receptor binding kinetics for both the fluorescent peptide ligand CHO-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine-fluorescein (CHO-NLFNTK-fl) and the non-fluorescent peptide ligand CHO-methionyl-leucyl-phenylalanine (CHO-MLF).

Actins↗
PubMed1998

Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase.

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.

Amino Acid Substitution↗