J. Ellis Bell

The lactose synthase acceptor site: a structural map derived from acceptor studies.

SummaryA pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either α or β-, but α- is somewhat preferred. In the absence of α-lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH2) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of α-lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (β-configuraation). However, upon α-lactalbumin binding the aglycone specificity allows for axial (α-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that α-lactalbumin binds to a hydrophobic site some distance from the C-2 group.

Kinetic properties and regulation of biliverdin reductase

In kinetic studies of the dual nucleotide enzyme biliverdin reductase, product inhibition patterns obtained with bilirubin as the inhibitor, using either the NADPH-linked reaction at pH 8.7 or the NADH-linked reaction at pH 7.0, are consistent with a random order of substrate addition and product release at either pH. With NAD(P) as the product inhibitor, the inhibition patterns suggest the existence of an enzyme-NAD(P)-biliverdin abortive complex. In the absence of added products, substrate inhibition observed at high biliverdin concentrations is also consistent with such a complex. Direct evidence for the existence of enzyme-NADP-biliverdin abortive complexes has been obtained at both pH 7.0 and 8.7 using the fluorescent NADP analog nicotinamide-1-N-6-ethenoadenine dinucleotide phosphate. Etheno-NADP fluorescence is enhanced in enzyme:etheno-NADP complexes but quenched in enzyme:biliverdin:etheno-NADP complexes. The effects of iron hematoporphyrin and cobalt protoporphyrin on the activity of biliverdin reductase have been examined. Iron-hematoporphyrin is a competitive inhibitor with respect to coenzyme at either pH. Comparison of Km values for biliverdin and Ki values for bilirubin or iron-hematoporphyrin indicate that while the binding of biliverdin is relatively pH independent, bilirubin binds more tightly at pH 7.0 than pH 8.7. Conversely, Fe-hematoporphyrin binds considerably tighter at pH 8.7 than pH 7.0. With cobalt-protoporphyrin, however, activation is observed, suggesting the existence of a regulatory site on biliverdin reductase for porphyrin derivatives. Resonance energy transfer measurements, from the fluorescent coenzyme analog etheno-NADP to biliverdin, iron-hematoporphyrin, or cobalt-protoporphyrin indicate that while the biliverdin site is located approximately 15 A from the etheno moiety of the coenzyme, both porphyrin derivatives are located 23.5 A away. This suggest the existence of a separate porphyrin binding site on biliverdin reductase which, depending upon the nature of the porphyrin binding, can produce activation or inhibition of the enzyme.

An exponential gradient maker for use with minigel polyacrylamide electrophoresis systems

Minigel electrophoresis has reduced the required sample size and cost of running polyacrylamide gels without loss of resolution. The many problems incurred in the minigel system when pouring multiple gradient gels are eliminated by pouring individual gels with the described economical, exponential gradient marker, which can accurately deal with the small gel volumes. The advantages of exponential gradients are discussed.

Investigation of the effects of crosslinking glutamate dehydrogenase with dimethyl pimelimidate

Chemical crosslinking with dimethyl pimelimidate has been used to examine the quaternary structure and conformational mobility of bovine liver glutamate dehydrogenase. Crosslinking patterns are shown to be consistent with either a stacked or staggered dimer of trimers structure of the hexamer. Crosslinking in the absence of coligands results in a small loss of activity but an almost complete loss of GTP inhibitory effects. Protection experiments show that the active site can be protected by a variety of ligand combinations, and that the loss of GTP inhibition is protected by several complexes containing either NADH or NADPH, indicating that the second coenzyme site per subunit (which preferentially binds NADH) is not involved in the protection process. A significant loss of ADP activation occurs during crosslinking which is not protected against by any combination of protecting ligands tried, including those which involve second coenzyme site binding, showing that the ADP site is functionally distinct from the GTP site and from the second coenzyme binding site. Crosslinking in the presence of protecting ligands gives similar gel patterns to those obtained in the absence of protection. Affinity chromatography experiments show that the crosslinked enzyme still binds GTP despite the loss of GTP inhibition, and hysteresis experiments show that the second coenzyme site is left functional if protected with either coenzyme. A model is presented where crosslinking affects the conformational linkage between various ligand binding sites involved in GTP inhibition rather than the sites themselves.

Glutamate dehydrogenase in Reye's syndrome Evidence for the presence of an altered enzyme in serum with increased susceptibility to inhibition by GTP

The glutamate dehydrogenase activity found in the serum of patients with Reyes syndrome is shown to be inhibited about 1000-fold more potently by GTP than is the normal human enzyme. 1 mM ADP, which with the normal enzyme effectively reverses GTP inhibition, has no effect in the GTP inhibition of the Reyes syndrome serum activity.

Interaction of terbium and calcium with chicken cystatin

The emission intensity of the fluorescent lanthanide, terbium, is shown to be enhanced upon binding to chicken cystatin. Fluorescence titrations indicate the presence of a single high affinity binding site per molecule. Binding of the terbium results in a 29% quenching of the fluorescence of the single tryptophan residue in the molecule. Calcium displaces the terbium from cystatin as judged by the decrease of terbium fluorescence in competition titrations. Similar titrations with magnesium or strontium demonstrate that the metal binding site of cystatin exhibits specificity for calcium or terbium. Analysis of the N-terminal sequence of chicken cystatin suggests the presence of a putative consensus sequence for a metal binding site between residues 13 and 24. Calcium causes a 17% decrease in the tryptophan fluorescence of cystatin, indicating that an induced conformational change accompanies metal binding. The increased quenching observed with terbium appears to be the result of resonance energy transfer from tryptophan to terbium. From the critical distance for energy transfer from tryptophan to terbium, it is estimated that the terbium binding site lies approximately 12 A from the single tryptophan residue in the molecule.

3,4,5,6-tetrahydrophthalic anhydride modification of glutamate dehydrogenase: The construction and activity of heterohexamers

Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.

Regulation of the immune response in the rat: Evidence for the existence of contrasuppressor cells☆

Abstract Rat splenocytes are shown to contain a population of plastic adherent, iron ingesting cells, presumably macrophages, which mediate suppression of concanavalin A-induced blastogenesis. A subpopulation of splenocytes which are separable from responding cells and suppressor macrophages by density gradient techniques has been obtained which negate the suppressive effects of macrophages. These cells, which appear to be a mature T-cell population, regulate the suppressive effects mediated by macrophages and are termed contrasuppressor cells.