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J E Porter

Publications and source records attributed to J E Porter.

35 records · Page 2Linked to original sources
PubMed1992

Effect of pH on subunit association and heat protection of soybean alpha-galactosidase.

Soybeans contain the enzyme alpha-galactosidase, which hydrolyzes alpha-1, 6 linkages in stachyose and raffinose to give sucrose and galactose. We have found that galactose, a competitive product inhibitor of alpha-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at pH 4.0 and at temperatures of up to 70 degrees C for 60 min. Stachyose and raffinose also protect alpha-galactosidase from denaturation at pH 4.0 although to a lesser extent. Glucose and mannose have little effect. At pH 7.0 the enzyme is a monomer, and galactose has no effect on the heat stability of the enzyme. In the absence of heat protection of the enzyme by added sugars, a series deactivation mechanism was found to describe the deactivation data. In comparison, a unimolecular, non-first order deactivation model applies at pH 4.0, where heat protection effects were observed. At a temperature above 60 degrees C, simple deactivation is a suitable model. The results suggest that alpha-galactosidase conformation and heat stability are directly related.

Enzyme Stability↗
PubMed1992

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.

Adenosine Triphosphatases↗
PubMed1992

Atypical alpha-1 adrenergic receptors on the rat parotid gland acinar cell.

Subtypes of alpha-1 adrenergic receptors on rat parotid gland acinar cell membranes were characterized using subtype selective alpha adrenergic receptor antagonists. The alpha-1 adrenergic receptor antagonist beta-iodo-[125I]-4-hydroxyphenyl-ethyl-aminomethyl-tetralone (125IBE) had an equilibrium dissociation constant for specific binding sites on these membranes of 0.241 +/- 0.03 nM and a total number of specific radioligand binding sites of 41 +/- 4 fmol bound/mg of protein. Displacement of 125IBE binding by subtype-selective alpha-1 adrenergic receptor antagonists 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane HCl (WB4101) and 5-methylurapidil fit best to biphasic competition curves. The high- and low-affinity inhibition equilibrium dissociation constant for WB4101 were 0.45 +/- 0.1 and 27 +/- 6 nM, respectively. Similarly, the high- and low-affinity inhibition equilibrium dissociation constants for 5-methylurapidil were 0.16 +/- 0.03 and 71 +/- 20 nM, respectively. These affinities for 125IBE binding sites suggest the presence of alpha-1A and alpha-1B adrenergic receptor subtypes on acinar cell membranes. The irreversible alpha-1 adrenergic receptor antagonist chloroethylclonidine was used to inactivate alpha-1B adrenergic receptors on acinar cell membranes. After treatment with chloroethylclonidine, saturation binding analysis demonstrated no change in the total number of 125IBE binding sites. In addition, competition curves for WB4101 and 5-methylurapidil again showed two sites of 125IBE displacement, with no change in antagonist affinities in membranes treated with chloroethylclonidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Calcium uptake in rat parotid gland secretory granules.

45Ca2+ uptake in isolated rat parotid secretory granules was examined in the presence of oxalate. Uptake of calcium was dependent on time, with the maximum occurring at 15 min. The uptake of calcium was dependent on adenosine-5'-triphosphate (ATP), and substitution of ATP with beta, gamma-methylene-ATP did not stimulate calcium uptake. Enzyme marker analysis indicated that mitochondria accounted for no greater than 3.0 +/- 0.2% of the observed ATP-dependent calcium uptake. Calcium uptake was blocked by the ATPase inhibitors tributyltin, IC50 = 12.2 +/- 0.6 nmol/L and 4-acetamido-4'-isothiocyano-2,2'-stilbene disulphonic acid (SITS), IC50 = 3.0 +/- 0.3 mumol/L. These results indicate that in the parotid secretory granule there is a calcium uptake mechanism that is dependent on the hydrolysis of ATP and is suppressed by two inhibitors of granule ATPase.

Adenosine Triphosphate↗
PubMed1990

Cost of a roller skating rink to the local accident and emergency department.

A 14 month retrospective study was undertaken to determine the cost implications of the opening of a roller skating rink to the local hospital accident and emergency department (A and E). A total of 398 patients attended following injury at the roller skating rink, of whom 384 were included in the study. The estimated cost of their injuries was determined by the hospital accounts department. The average cost per patient attending the A and E department following roller skating injury was about 100 pounds. The total cost to the A and E department of all injuries sustained at the rink over this period was 38,412 pounds. The cost implications of opening a roller skating rink for the A and E department are considerable. If proposals for self-budgeting are applied, A and E departments will have to seek additional funding if such leisure facilities are opened in their vicinity.

Adolescent↗
PubMedDate not supplied

Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C.

A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.

Animals↗
PubMedDate not supplied

Optimization of ion-exchange protein separations using a vector quantizing neural network.

In this work, a previously proposed methodology for the optimization of analytical scale protein separations using ion-exchange chromatography is subjected to two challenging case studies. The optimization methodology uses a Doehlert shell design for design of experiments and a novel criteria function to rank chromatograms in order of desirability. This chromatographic optimization function (COF) accounts for the separation between neighboring peaks, the total number of peaks eluted, and total analysis time. The COF is penalized when undesirable peak geometries (i.e., skewed and/or shouldered peaks) are present as determined by a vector quantizing neural network. Results of the COF analysis are fit to a quadratic response model, which is optimized with respect to the optimization variables using an advanced Nelder and Mead simplex algorithm. The optimization methodology is tested on two case study sample mixtures, the first of which is composed of equal parts of lysozyme, conalbumin, bovine serum albumin, and transferrin, and the second of which contains equal parts of conalbumin, bovine serum albumin, tranferrin, beta-lactoglobulin, insulin, and alpha -chymotrypsinogen A. Mobile-phase pH and gradient length are optimized to achieve baseline resolution of all solutes for both case studies in acceptably short analysis times, thus demonstrating the usefulness of the empirical optimization methodology.

Algorithms↗