Biomedical subjects

S P Brooks

Publications and source records attributed to S P Brooks.

At least 19 recordsLinked to original sources

PubMed1995

A comparison of methods for determining total body protein.

The aim of the study was to find the optimal method (with respect to convenience and accuracy) for determining total protein in whole-body homogenates of rats. Three different protein extraction methods and five different protein concentration methods were assessed. The results were compared against a reference value measured by complete amino acid analysis after acid hydrolysis. The data demonstrated that extraction with 5% (w/v) sodium dodecylsulfate (SDS) in 0.5 N NaOH was far superior to that with water alone or to 6 N guanidine-HCl. A comparison of the Biuret, Bradford, and bicinchoninic acid methods on the SDS-NaOH-extracted samples showed that the Biuret method was optimal, giving a value that was 90% of the reference value with a small variation (2.4% of the mean). The Kjeldahl method gave the correct protein concentration only when a nitrogen factor of 5.51 +/- 0.03 (N = 5) was applied. The results suggest that extraction with SDS-NaOH followed by the Biuret procedure is a good method for measuring protein concentrations in whole body rat homogenates.

Amino Acids↗

PubMed1995

Protein phosphorylation patterns during aestivation in the land snail Otala lactea.

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivating Otala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of 32P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7-10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60-80 kDa and 30-40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30-35 kDa and 40-45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16-22 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Evidence for aestivation specific proteins in Otala lactea.

Changes in [35S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails, Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest that O. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.

Animals↗

PubMed1995

Activation of multiple transcription factors and fos and jun gene family expression in cells exposed to a single electric pulse.

We report that exposure of cells to a single electric pulse (250-1250 V/cm) results in the rapid and persistent activation of the DNA binding activities of a number of transcription factors, including AP-1, SP1, AP-2, and NF-kappa B, and the transient expression of select members of the fos and jun gene families. Induction of gene expression occurs primarily at the level of transcription, although c-jun expression also appears to be regulated posttranscriptionally. Interestingly, maximal induction of gene expression is detected at electrical field strengths that do not result in pore formation in the plasma membrane and that do not significantly affect cell viability. Exposure of cells to electric pulses does not result in the activation of HSF1 DNA binding activity, or the induction of hsp70 or p53 protein synthesis, indicating that the induction of fos and jun gene expression is not coincident with protein or DNA damage. The results of these studies suggest that electrical pulses may represent a novel mechanism for inducing the activities of multiple transcription factors and the expression of select members of the fos and jun gene families.

3T3 Cells↗

PubMed1994

Metabolic depression in land snails: in vitro analysis of protein kinase involvement in pyruvate kinase control in isolated Otala lactea tissues.

Isolated tissues from the land snail Otala lactea were used to examine the relationship between protein kinase activity and phosphorylation-induced changes associated with metabolic depression. Hepatopancreas and foot muscle were removed from active and estivating land snails and incubated in vitro under aerobic and anoxic conditions. Pyruvate kinase (PK), cAMP-dependent protein kinase (PKA), and protein kinase second messenger compounds (cyclic AMP and inositol 1,4,5-triphosphate) were measured after incubating the tissues for 4 hours. Pyruvate kinase from the hepatopancreas of active snails was phosphorylated during anoxic incubations as indicated by changes in the I50 value for L-alanine. However, measurements of PKA activity and of cellular cAMP concentrations suggested that PKA activity was lower in these incubated tissues. When foot muscle was used as the tissue source, incubation under anoxic conditions produced no changes in PK activity even though PKA activity was drastically reduced. Analysis of changes in inositol 1,4,5-triphosphate concentrations after tissue incubation showed that they were not consistent with changes in PK activity in either organ. These results suggest that PKA and Ca2+/phospholipid-dependent protein kinase C do not phosphorylate PK during anoxia in land snails. The differences between values measured in incubated tissues and those measured in vivo suggest that isolated O. lactea tissues are not a good in vitro model system for studying metabolic changes associated with depressed metabolism.

Animals↗

PubMed1994

Effect of anoxia on isolated turtle tissues: is the response to anoxia mediated by protein kinase second messengers?

Organ slices from the turtle Trachemys scripta elegans were incubated under aerobic and anoxic conditions to examine the effect of protein kinase (PrK) second messengers in potentiating the biochemical responses to anoxia exposure. Incubating liver slices from aerobic animals under anoxic conditions produced biochemical changes exactly similar to those observed in vivo: phosphofructokinase (PFK) was more sensitive to citrate inhibition and the percentage of glycogen phosphorylase (GP) in the active a form increased. On the other hand, incubating brain and heart tissue slices under anoxic conditions produced no changes in PFK and GP kinetic constants. Addition of PrK second messengers (dibutyryl-cAMP or Ca2+ plus phorbol myristate acetate) to the incubated tissues did not promote anoxia-associated changes in aerobically incubated tissues nor did they prevent anoxia-associated changes in anaerobically incubated tissues. These results suggest that unidentified external hormonal signals mediate heart and brain responses to anoxia. It is also apparent that cAMP and Ca2+ plus phospholipid do not play a role in bringing about the anoxia-induced changes in PFK, GP and fructose 2,6-bisphosphate in liver of turtles.

Aerobiosis↗

PubMed1994

A program for analyzing enzyme rate data obtained from a microplate reader.

A computer program (microplate analysis [MPA] program) is described that allows manipulation of kinetic data obtained from a microplate reader. It is particularly useful for obtaining enzyme velocities from microplate data. Data can be displayed either as an 8 x 12 grid which shows all microplate wells at low resolution or as single enzyme progress curves which show the reaction time course in greater detail and allow data manipulation (single curve mode). In the single curve mode, the X-axis of the progress curves can be displayed either as discrete time points numbered from the first to the last or as real time in seconds. The program can calculate the maximum, minimum or initial reaction rate by either linear or robust regression. In the single curve mode, the user can control the range of data points included in the regression analysis and the number of data points over which the rate is averaged. In this way, the maximum (or minimum) rate within a specific range of data can be determined. The major advantage of the MPA program is that the range settings are specific to each well. This gives the user complete control over the presentation and calculation of the kinetic data. The MPA program requires an AS-CII file containing data blocks of 96 sequential absorbance readings for each time point and the time interval between successive absorbance readings.

Calibration↗

PubMed1993

An improvement in the pyruvate dehydrogenase complex assay: a high-yield method for purifying arylamine acetyltransferase.

A new method for purifying arylamine acetyltransferase (AAT) has been devised using polyethylene glycol fractionation and hydroxylapatite chromatography. The new procedure gives a final yield of approximately 70% based on activity in crude homogenates and can be performed in a single day. This represents a threefold higher yield than previous methods. The procedure may be used to purify AAT from pigeon, chicken, and duck livers with equivalent yield of the final enzyme. However, the preparation from pigeon liver is preferred because of the sevenfold higher activity in this tissue. Nevertheless, if fresh pigeon livers cannot be acquired, calculations reveal that the preparation from chicken liver is comparable to that from pigeon liver acetone powder with respect to total activity obtained per gram wet weight starting material. One can also calculate that the chicken liver preparation is approximately 40 times cheaper than that from pigeon liver acetone powder, making the preparation of AAT from fresh chicken livers a good alternative when pigeon livers are not available.

Aniline Compounds↗

PubMed1993

Control of glycolytic enzyme binding: effect of changing enzyme substrate concentrations on in vivo enzyme distributions.

The effect of changing concentrations of glycolytic intermediates on the binding of phosphofructokinase, aldolase and pyruvate kinase to cellular particulate matter was investigated. Concentrations of glycolytic intermediates were altered by adding 2 mM iodoacetic acid (IAA) to an incubation medium containing tissues isolated from the channelled whelk Busycon canaliculatum. Iodoacetic acid inhibited glyceraldehyde 3-phosphate dehydrogenase activity causing a 100-400 fold increase in the concentration of fructose 1,6-bisphosphate as well as 3-20 fold increases in glucose 6-phosphate, fructose 6-phosphate, and dihydroxyacetone phosphate levels depending on the experimental protocol. Cellular pH values were not statistically different in the presence of IAA. Measurement of enzyme binding to particulate matter showed that the binding of phosphofructokinase, aldolase and pyruvate kinase was unaffected by iodoacetic acid under any experimental condition. These results show that changes in the tissue concentrations of enzyme substrates and products do not regulate enzyme binding to particulate matter in the cell.

Animals↗

PubMed1993

De novo protein synthesis and protein phosphorylation during anoxia and recovery in the red-eared turtle.

Changes in de novo protein synthesis and protein phosphorylation were monitored during anoxia and recovery in the red-eared slider Trachemys (= Pseudemys) scripta elegans. Time courses of 35S-radiolabeled methionine incorporation into acid-precipitable material showed an increase up to 5 h postinjection and remained constant after this time. Comparison of the total and acid-precipitable 35S label incorporation into tissues from 20-h control, anoxic, and recovering animals showed differences between these groups: total radioactivity in brain was 2.9-fold lower in recovering turtles, whereas protein-associated radioactivity was 2.4-fold higher in anoxic liver, 2.3-fold lower in recovering skeletal muscle, and 3.7-fold lower in recovering brain tissue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled proteins showed the existence of a newly synthesized protein band (relative molecular mass = 72 kDa) that was apparent only in 20-h recovering liver and skeletal muscle. Use of 32P labeling to monitor changes in protein phosphorylation patterns during anoxia revealed 1.6-, 1.4-, and 1.5-fold increases in 32P incorporation in anoxic brain, heart, and liver, respectively. Changes in protein phosphorylation were localized to the plasma membrane and cytosolic fractions in brain and to the cytosolic fraction in liver.

Animals↗

PubMed1992

A kinetic description of sequential, reversible, Michaelis-Menten reactions: practical application of theory to metabolic pathways.

Equations are presented which describe a linear coupled system of reactions that utilize a single substrate and convert it to product by way of several intermediate enzyme catalysed steps. The present analysis extends previous results by assuming that the enzymes obey reversible Michaelis-Menten kinetics. In order for the system to reach steady state one must assume that the initial substrate concentration and the final product concentration are buffered to a constant value. Using the present analysis it can be shown that the system will not enter a steady state if the maximal velocity of any forward reaction is less than the steady state flux through the system. This condition represents a practical test for determining if a system will enter steady state but is valid only when the rate of the primary enzyme is not affected allosterically be intermediates in the pathway. The equations are used to analyse a portion of the rat liver glycogenic pathway that catalyses the conversion of glucose to fructose 1,6-bisphosphate.

Animals↗

PubMed1992

Bound and determined: a computer program for making buffers of defined ion concentrations.

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40 degrees C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i-iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.

Adenosine Triphosphate↗

PubMed1992

A simple computer program with statistical tests for the analysis of enzyme kinetics.

A simple computer program that calculates the kinetic parameters of enzyme reactions is described. Parameters are determined by nonlinear, least-squares regression using either Marquardt-Levenberg or Gauss-Newton algorithms to find the minimum sum of squares. Three types of enzyme reactions can be analyzed: single substrate reactions (Michaelis-Menten and sigmoidal kinetics), enzyme activation at a fixed substrate value or enzyme inhibition at a fixed substrate value. The user can monitor goodness of fit through nonparametric statistical tests (performed automatically by the computer) and through visual examination of the pattern of residuals. The program is unique in providing equations for activator and inhibition analysis as well as in enabling the user to fix some of the parameters before regression analysis. The simplicity of the program makes it extremely useful for quickly determining kinetic parameters during the data-gathering process.

Biometry↗

PubMed1991

Re-evaluation of the glycerol-3-phosphate dehydrogenase/L-lactate dehydrogenase enzyme system. Evidence against the direct transfer of NADH between active sites.

An investigation of the direct transfer of metabolites from rabbit muscle L-lactate dehydrogenase (LDH, EC 1.1.1.27) to glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) revealed discrepancies between theoretical predictions and experimental results. Measurements of the GPDH reaction rate at a fixed NADH concentration and in the presence of increasing LDH concentrations gave experimental results similar to those previously obtained by Srivastava, Smolen, Betts, Fukushima, Spivey & Bernhard [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6464-6468]. However, a mathematical solution of the direct-transfer-mechanism equations as described by Srivastava et al. (1989) showed that the direct-transfer model did not adequately describe the experimental behaviour of the reaction rate at increasing LDH concentrations. In addition, experiments designed to measure the formation of an LDH4.NADH.GPDH2 complex, predicted by the direct-transfer model, indicated that no significant formation of tertiary complex occurred. An examination of other kinetic models, developed to describe the LDH/GPDH/NADH system better, revealed that the experimental results may be best explained by assuming that free NADH, and not E1.NADH, is the sole substrate for GPDH. These results suggest that direct transfer of NADH between rabbit muscle LDH and GPDH does not occur in vitro.

Animals↗

PubMed1991

A quantitative evaluation of the effect of enzyme complexes on the glycolytic rate in vivo: mathematical modeling of the glycolytic complex.

The cellular distribution of free and bound glycolytic enzymes in vivo was estimated by means of a model based on previously determined association constants for individual binding interactions and in vivo protein concentrations. The calculations revealed that a significant proportion of the enzymes would be either associated with F-actin, or bound in binary enzyme-enzyme complexes in vivo. An analysis of the relative concentration, and relative activity, of F-actin-bound enzymes suggested that a complete glycolytic complex, composed of all enzymatic steps from phosphofructokinase (PFK) to lactate dehydrogenase (LDH) does not exist. This was indicated by a very low concentration of F-actin-associated phosphoglycerate kinase (PGK) and by a very low activity of F-actin bound aldolase and PGK; this model showed that aldolase and PGK would be absent from any F-actin bound complex. An analysis of soluble enzyme-enzyme associations indicated that formation of binary enzyme complexes may lead to an increased overall flux through glyceraldehyde 3-phosphate dehydrogenase and LDH, but would serve to decrease flux through PFK and aldolase. A 1.4-fold activation of PFK, which occurs when the soluble enzyme binds to F-actin, suggested that reversible binding of PFK to F-actin may represent a novel cellular mechanism for controlling glycolytic flux during periods of increased metabolic demand by controlling the key regulatory enzyme of glycolysis.

Actins↗

PubMed1991

The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase.

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.

Adenosine Triphosphate↗

PubMed1991

Where is the glycolytic complex? A critical evaluation of present data from muscle tissue.

Associations between glycolytic enzymes and subcellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzyme binding to subcellular structural components is believed to regulate enzyme activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that enzyme binding to cellular structures is not involved in the control of glycolysis. (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strength and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist. (ii) In many cases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity. (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negligible.

Actins↗

PubMed1991

Enhanced resection and improved survival in murine neuroblastoma (C1300-NB) after preoperative immunotherapy.

Advanced neuroblastoma treated with standard chemotherapy has a poor prognosis. Combination immunotherapy for murine neuroblastoma with retinyl palmitate, low-dose cyclophosphamide, and interleukin-2 resulted in increased survival, impaired tumor growth, easier surgical resection, and increased class I expression or tumor cells. Preoperative immunotherapy may be useful in treatment of advanced human neuroblastoma.

Animals↗