Apparent Substrate Affinity Research Articles

Ligand Recognition by the Vesicular Monoamine Transporters

Molecular cloning has identified three distinct proteins responsible for plasma membrane monoamine transport. Although functional analysis indicates that each can recognize multiple monoamines as substrates, selective expression within dopamine, norepinephrine, and serotonin cell populations of the brain suggests that each probably transports a single substrate in vivo . In contrast, molecular cloning has identified two vesicular monoamine transporters (VMATs), one expressed in the adrenal medulla and other neuroendocrine tissues (VMAT1) and the other in the brain (VMAT2). Although VMATs have no sequence similarity to the plasma membrane transporters or receptors for monoamines, they do contain individual residues implicated in substrate recognition by these other proteins. In addition to those residues required for substrate recognition, the comparison of VMAT1 and VMAT2 indicates that other residues may modulate the recognition of ligand. VMAT2 has an apparent affinity for most substrates approximately threefold higher than VMAT1. In addition, only VMAT2 recognizes histamine, and tetrabenazine inhibits only VMAT2. Residues have been identified that have a variety of roles in substrate recognition by VMATs. Analogous to other membrane proteins that interact with monoamines, VMATs contain an aspartate in TMD1 and serines in TMD3 that are required for transport activity.

Effects of Conserved Residues on the Regulation of Rabbit Muscle Pyruvate Kinase

A cDNA encoding the complete rabbit muscle pyruvate kinase isozyme (RMPK) was cloned using the method of rapid amplification of cDNA ends. The sequence encodes a polypeptide chain of 530 amino acids which differs in three amino acid residues from a sequence reported by Larsen et al. (Larsen, T.M., Laughlin, T., Holden, H.M., Rayment, L, and Reed, G.H. (1994) Biochemistry 33, 6301-6309). Glu233-Gln234 and Ala400 were identified instead of Asp233-Glu234 and Ser400, respectively. The recombinant RMPK was overexpressed in the Escherichia coli JM 105 cells. Purified recombinant pyruvate kinase displayed identical physical and enzymatic properties as the authentic enzyme. Three point mutants of RMPK were constructed using site-directed mutagenesis. Like the wild type RMPK, sedimentation, and CD spectroscopic studies show that purified RI 19C and T340M are tetrameric proteins with similar secondary and tertiary structures. Mutant R119C enzyme exhibits 0.6% of the value of k(cat) and an order of magnitude decrease in the apparent affinity for ADP as compared to the wild type PK. The overall response to inhibitor and activator, Phe and FBP, respectively, were not affected by the R119C mutation. The T340M mutant enzyme is only half as active as the wild type PK. T340M is more susceptible to inhibition by Phe but apparently is not responsive to the activator FBP. The kinetic behavior of the Q377K mutant enzyme is in between that of the R119C and T340M mutants exhibiting 5% of the wild type enzymatic activity and an enhanced sensitivity to the inhibitor, Phe, while maintaining the same responsiveness to FBP and apparent affinities for substrates. The significant decrease in activity in all three mutants mimics the exact consequences of the same mutations in human erythrocyte PK from hemolytic anemia patients. Thus, this study demonstrates not only the effects of these conserved residues in the regulatory properties of mammalian PK. but also that the observed effects are most likely applicable to all isozymic forms of PK.

Chimeric Vesicular Monoamine Transporters Identify Structural Domains That Influence Substrate Affinity and Sensitivity to Tetrabenazine

The vesicular monoamine transporters (VMATs) 1 and 2 show close sequence similarity but substantial differences in apparent substrate affinity and drug sensitivity. To identify structural domains that determine these functional characteristics, chimeric transporters were constructed and their properties were analyzed in a heterologous expression system. The results implicate multiple regions in the recognition of serotonin and histamine and the sensitivity to tetrabenazine. Two domains of VMAT2, one extending from transmembrane domain (TMD) 5 to the beginning of TMD8 and the other from the end of TMD9 through TMD12, increase the affinity for serotonin and histamine as well as the sensitivity to tetrabenazine but only in the context of more C-terminal and more N-terminal VMAT2 sequences, respectively. In addition, the extreme N terminus of VMAT2 alone suffices to confer a partial increase in substrate affinity and tetrabenazine sensitivity. Despite these similarities among the interactions with serotonin, histamine, and tetrabenazine, the region of VMAT2 from TMD3 through TMD4 increases serotonin affinity but not histamine affinity or tetrabenazine sensitivity, and whereas the region from TMD5 to TMD8 of VMAT2 increases serotonin affinity in the context of more C-terminal VMAT2 sequences, the region encompassing TMD5 through TMD7 reduces serotonin but not histamine affinity or tetrabenazine sensitivity in the context of more N-terminal VMAT2 sequences. Thus, the chimeric analysis also reveals differences between serotonin recognition and the recognition of both histamine and tetrabenazine that may account for the observed differences in their interaction with the transport protein.

Calcium channel antagonist verapamil modulates human spermatozoal functions.

The effect of calcium channel blocker, verapamil (0.5-50 microM), has been studied in vitro in relation to certain spermatozoal functions in human ejaculates. Disruptive changes in the head and tail region of the spermatozoa, separation of heads from tails and coiling of the tail were observed. Motility was considerably reduced, while the pattern of motility also changed from rapid, linear progression to slow or sluggish linear or non-linear movement and finally to non-progressive motility, or even immotility. Verapamil significantly inhibited the influx of extracellular Ca2+. The study of kinetic effects further revealed a reduction in the maximum uptake velocity, but no change in the apparent substrate affinity constant. A highly significant decrease in Ca(2+)-dependent ATPase activity was also noted. In order to see whether this drug had any cytotoxic effect, presumably through lipid peroxides, thiobarbituric acid-reactive substances were measured. Verapamil produced an increase in the formation and release of malonyldialdehyde. The level of membrane cholesterol and phospholipid in the spermatozoa was also lowered considerably. The potential of such a calcium channel blocking agent in the designing of a male contraceptive programme is discussed.

L-Alanine transport in small intestine brush-border membrane vesicles of obese rats

Membrane vesicles from the small intestine brush border were obtained and used to determine the possible effects of genetic or nutritional obesity on l-alanine uptake. Membrane vesicles from Zucker fa fa obese rats and cafeteria diet-fed Zucker Fa/? rats showed the same characteristics as those of standard diet-fed lean animals. All preparations showed sodium-dependent transport as the main pathway for l-alanine uptake within the substrate concentration range tested. The apparent substrate affinity constant ( K rmm ) values and the pattern of inhibition of Na +-dependent l-alanine uptake by other amino acids ( l-leucine and l-glutamine), suggests that system B involved in the transport of dipolar amino acids (formerly named Neutral Brush Border System) participates in the Na +-dependent transport of l-alanine. The affinity constant ( K m) for l-alanine was essentially the same for all the groups studied (in the range of 10 mM). However, there was a higher ( P < 0.05) maximal capacity ( V max) in preparations from diet-induced obese animals (cafeteria diet) than that of genetically obese rats. These results indicate that either nutritional or genetic obesity may modify the capacity but not the affinity of transport systems for l-alanine uptake in the brush border of rat small intestine.

Aromatase Activity in Developing Guinea Pig Brain: Ontogeny and Effects of Exogenous Androgens1

The formation of estrogens from androgens by aromatase in the developing brain is an important step in the sexual differentiation of many species. We characterized aromatase activity (AA) in a high-speed pellet of brain tissue from fetal guinea pigs. The apparent substrate affinity (approximately 17 nM) was comparable to reported values in other species. Aromatase activity was highest in the amygdala (AMG) and preoptic area (POA), with lesser amounts in the septum (SEPT) and medial basal hypothalamus (MBH). Activity was low but measurable in parietal cortex (CTX). In the AMG, POA, SEPT, and MBH, AA was highest in early gestation (Days 35-40) and showed a steady decline through development. No sex difference in AA was apparent. We also determined the effects of administration of exogenous androgens to pregnant females on brain AA in the fetus. Testosterone propionate (5 mg/day on Days 30-39 followed by 1 mg/day on Days 40-50) caused a significant increase (p < 0.05) in AA found in the MBH and CTX. Administration of dihydrotestosterone propionate (2.5 mg/day on Days 30-39 followed by 1 mg/day on Days 40-50) significantly stimulated AA in SEPT, MBH, and CTX. These data demonstrate that the fetal guinea pig brain contains high levels of AA during the critical period of sexual differentiation. Treatment with high levels of exogenous androgens consistently induces AA in the MBH and CTX. These latter effects may be among the mechanisms through which exogenous androgens act on the developing brain.

Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in human platelets.

Receptor-mediated activation of human platelets by thrombin initiates a series of rapid biochemical events that include activation of phospholipase A2 to liberate arachidonic acid for further conversion to thromboxane A2. The identity of the phospholipase A2 involved has not been clear. Here we show by immunochemical analysis that human platelets contain significant amounts (60 ng/10(9) platelets) of the recently identified Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2). Metabolic labeling of human platelets with 33Pi revealed that the extent of phosphorylation of cPLA2 was greatly increased after thrombin treatment. Upon stimulation of platelets with thrombin, cPLA2 exhibits enhanced catalytic activity, as well as a change in its electrophoretic and chromatographic properties compared with cPLA2 in resting platelets. These alterations of cPLA2 are reversed by treatment with phosphatase, demonstrating that they are the consequence of thrombin-stimulated phosphorylation. Thrombin-induced phosphorylation and activation of cPLA2 is rapid (half-maximal by 1 min at 1 unit/10(9) platelets) and dose-dependent. Agonist-induced phosphorylation of cPLA2 is more sensitive to thrombin than the generation of thromboxane A2, suggesting that it may be an early event in the sequence of steps leading to the mobilization and further metabolism of arachidonic acid. By comparing the functional properties of cPLA2 from control versus thrombin-stimulated platelets, we found that while activated cPLA2 exhibits the same Ca2+ requirement and apparent substrate affinity (Km), its catalytic activity (Vmax) is increased compared with control cPLA2. We conclude that 1) cPLA2 is likely to play an important role in agonist-induced mobilization of arachidonic acid and 2) thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca2+ but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity.

Cloning, Expression, and Sequence of an Allosteric Mutant ADPglucose Pyrophosphorylase from Escherichia coli B

Escherichia coli B mutant strain SGL4 accumulates glycogen at 28% of the rate observed for the parent strain. This is due to the presence of an ADPglucose pyrophosphorylase with altered allosteric properties including lower apparent affinities for substrates, the activator, fructose-1,6-bisphosphate, and the inhibitor, AMP. The mutant enzyme also is completely insensitive to activation by NADPH. To clone this mutant, an SG14 library was constructed by insertion of the chromosomal DNA into the PstI site of pBR322. Screening of the library via colony hybridization with a wild-type gene (glgC) probe resulted in the successful isolation of a recombinant plasmid, designated pPJ2, which contained the mutant glgC gene. The enzyme expressed from pPJ2 was partially purified and found to be very similar in kinetic and allosteric properties to the enzyme isolated from the SG14 strain. The mutant glgC gene, a HincII fragment from pPJ2, was then subcloned into pUC118/119 for dideoxy sequencing of both strands. One amino acid change was found in a region that is highly conserved in all known sequences: a single point mutation at the deduced amino acid residue 44 resulted in a change of alanine to threonine. The properties of this mutant are discussed in comparison to other known allosteric mutants.

Stage-related changes in steroid-converting enzyme activities in Squalus testis: synthesis of biologically active metabolites via 3β-hydroxysteroid dehydrogenase/ isomerase and 5α-reductase

To investigate the relationship between steroidogenesis and spermatogenesis, two key enzymes of androgen biosynthesis, 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and 5α-reductase, were compared at premeiotic (PrM), meiotic (M) and postmeiotic (PoM) stages. Staged tissues were obtained by dissection from the testis of the spiny dogfish Squalus acanthias, in which spermatogenesis is a simple diametric progression. Microsomal 3β-HSD activity was measured by conversion of [ 3H] dehydroepiandrosterone (DHEA) to androstenedione (AE). Reaction constants were: K m = 3.2 μM and V max = 243 pmol/min/mg protein. 3β-HSD increased progressively with maturation, resulting in three- to four-fold higher levels in PoM than in PrM stages. Absolute values and stage-related differences were the same, whether microsomes were derived from whole testis or from isolated spermatocysts (germ cell/Sertoli cell units), thus supporting microscopic studies showing that Sertoli cells are the primary steroidogenic elements of dogfish testis. In vitro conversion of [ 3H]testosterone to [ 3H]dihydrotesterone (DHT) was used to estimated 5α-reductase activity. Apparent substrate affinity was similar to that of 3β-HSD ( K m = 2.9 μM), but maximal product yields were two to three orders of magnitude lower ( V max = 208 fmol/min/mg protein). Also, the stage-related pattern of 5α-reductase activity ( PrM > PoM >> M) differed from that of 3β-HSD ( PoM >> M > PrM). Together with previously measured steroidogenic enzyme activities using the shark testis model, these results predict that de novo androgen biosynthesis is highest in testicular regions with mature spermatids and fully differentiated Sertoli cells, and also suggest that availability of T versus DHT for androgen receptor binding may be developmentally regulated during spermatogenesis.

Rapid Modulation of Spinach Leaf Nitrate Reductase Activity by Photosynthesis

It has been shown recently that in spinach leaves (Spinacia oleracea) net photosynthesis and nitrate reduction are closely linked: when net photosynthesis was low because of stomatal closure, rates of nitrate reduction decreased (WM Kaiser, J Förster [1989] Plant Physiol 91: 970-974). Here we present evidence that photosynthesis regulates nitrate reduction by modulating nitrate reductase activity (NRA, EC 1.6.6.1). When spinach leaves were exposed to low CO(2) in the light, extractable NRA declined rapidly with a half-time of 15 minutes. The inhibition was rapidly reversed when leaves were brought back to air. NRA was also inhibited when leaves were wilted in air; this inhibition was due to decreased CO(2) supply as a consequence of stomatal closure. The modulation of NRA was stable in vitro. It was not reversed by gel filtration. In contrast, the in vitro inhibition of nitrate reductase (NR) by classical inhibitors such as cyanide, hydroxylamin, or NADH disappeared after removal of free inhibitors by gel filtration. The negative modulation of NRA in -CO(2)-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg(2+) or Ca(2+). Mg(2+) concentrations required for observing half-maximal inhibition were about 1 millimolar. In the presence of EDTA, the enzyme activity was always high and rather independent of the activation status of the enzyme. NRA was also independent of the pH in the range from pH 7 to pH 8, at saturating substrate and Mg(2+) concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only V(max) changed.

Purification and Characterization of Phosphoglycolate Phosphatase from the Cyanobacterium Coccochloris peniocystis

The properties and role of the enzyme phosphoglycolate phosphatase in the cyanobacterium Coccochloris peniocystis have been investigated. Phosphoglycolate phosphatase was purified 92-fold and had a native molecular mass of approximately 56 kilodaltons. The enzyme demonstrated a broad pH optimum of pH 5.0 to 7.5 and showed a relatively low apparent affinity for substrate (K(m) = 222 micromolar) when compared to that from higher plants. The enzyme required both an anion and divalent cation for activity. Mn(2+) and Mg(2+) were effective divalent cations while Cl(-) was the most effective anion tested. The enzyme was specific for phosphoglycolate and did not show any activity toward a variety of organic phosphate esters. Growth of the cells on high CO(2) and transfer to air did not result in any significant change in phosphoglycolate phosphatase activity. Competitive inhibition of C. peniocystis triose phosphate isomerase by phosphoglycolate was demonstrated (K(i) = 12.9 micromolar). These results indicate the presence of a specific noninducible phosphoglycolate phosphatase whose sole function may be to hydrolyze phosphoglycolate and prevent phosphoglycolate inhibition of triose phosphate isomerase.

Aromatase Activity in Adult Guinea Pig Brain is Androgen Dependent1

Androgen metabolism in target tissues constitutes an important step for understanding hormone action. The in situ aromatization of androgen represents one of these metabolic events. We characterized aromatase activity (AA) in a microsomal preparation of brain tissue from adult guinea pigs since earlier reports questioned its presence in neural tissues of this species. Analyses revealed an apparent substrate affinity (approximately 17 nM) that was equivalent in adult males and females. However, adult male brains contained greater quantities of AA than female brains. Specifically, AA in the preoptic area (POA: p less than 0.05) and the medial basal hypothalamus (MBH; p less than 0.01) was greater in males than in females. AA was concentrated in the limbic system and hypothalamus (amygdala greater than POA greater than septum greater than MBH), whereas low levels were consistently measured in cortical tissue. In vitro estrogen formation was significantly lower in POA (p less than 0.05) and MBH (p less than 0.01) after castration. After dihydrotestosterone treatment, AA returned to levels equal to or greater than those observed in intact males. These data indicate that AA does exist in the guinea pig brain and is modulated by androgens through the androgen receptor. The presence of high levels of aromatase activity may suggest a role for locally formed estrogens in brain function in this species.

Angiotensin II stimulation of hydrogen ion secretion in the rat early proximal tubule. Modes of action, mechanism, and kinetics.

Physiologic concentrations of angiotensin II stimulate sodium transport by intestinal and renal early (S1) and late (S2) proximal tubule epithelial cells. We recently found that hydrogen ion secretion, which effects sodium bicarbonate absorption, was a transport function preferentially and potently increased by angiotensin II in S1 cells. S1 cells are normally responsible for half of the total renal hydrogen ion secretion. The mechanism by which angiotensin II regulates intestinal sodium transport is by potentiating sympathetic nerve activity and norepinephrine release. Direct control of hydrogen ion secretion by angiotensin II via receptors on epithelial cells has not been previously demonstrated. We now report that stimulation of in vivo hydrogen ion secretion in the rat early proximal tubule by angiotensin II was not mediated via change in nerve activity. Rather, enhanced hydrogen ion secretion by angiotensin II correlated with increased angiotensin II receptor density on epithelial cells in the early compared to late microdissected proximal tubule. Basolateral as well as luminal angiotensin II stimulated bicarbonate absorption. Angiotensin II reduced bicarbonate permeability and caused alteration in the apparent substrate affinity, but not maximal capacity, of the proximal hydrogen ion secretory system involving the Na+/H+ antiporter.

Buffer, pH, and ionic strength effects on the ( Na+ + K+)-ATPase

A dog kidney ( Na + + K +)-ATPase preparation also catalyzes K +-independent and K +-activated phosphatase reactions with p-nitrophenyl phosphate as substrate. K +-independent activity increases with declining pH over the range 7.5 to 5.8, whereas the other two activities decrease. The increased K +-independent activity is similar with imidazole, histidine, and several Good buffers, and is thus attributable to free H +, probably by affecting enzyme conformations rather than by changing affinity for Mg 2+ or substrate or by H + occupying specific K +-sites. The decrease in K +-phosphatase and (Na + + K +)-ATPase activities with pH also occurs similarly with those buffers, and is not due to changes in apparent affinity for substrate or for cation activators. However, the Good buffers Pipes and ADA inhibit the K +-independent phosphatase reaction strongly, the K +-activated reaction moderately, and the (Na + + K +)-ATPase reaction little; both contain two acidic groups, unlike the other buffers tested. Inhibition of the phosphatase reaction by Pipes is associated with a decreased apparent affinity for K + and an increased sensitivity to inhibition by Na + and ADP, consistent with Pipes hindering conformational transitions to the E 2 enzyme forms required for phosphatase hydrolytic activity.

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady-state kinetic properties

Dihydropyrimidine amidohydrolase (EC 3.5.2.2) catalyzes the reversible hydrolysis of 5,6-dihydropyrimidines to the corresponding β-ureido acids. Previous work has shown that incubation of this Zn 2+ metalloenzyme with 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline results in inactivation by Zn 2+ removal by a reaction pathway involving formation of a ternary enzyme-Zn 2+-chelator complex which subsequently dissociates to yield apoenzyme and the Zn 2+-chelate (K. P. Brooks, E. A. Jones, B. D. Kim, and E. G. Sander, (1983) Arch. Biochem. Biophys. 226,469–483). In the present work, the pH dependence of chelator inactivation is studied. The equilibrium constant for formation of the ternary complex is strongly pH dependent and increases with decreasing pH for all three chelators. There is a positive correlation between the value of the equilibrium constant observed for each chelator and the value of its stability constant for formation of Zn 2+-chelate. The affinity of the chelators for the enzyme increases in the order 8-hydroxyquinoline-5-sulfonic acid > o-phenanthroline > 2,6 dipicolinic acid. The first-order rate constant for breakdown of the ternary complex to yield apoenzyme and Zn 2+-chelate is invariant with pH for a given chelator but is different for each chelator, increasing in the reverse order. The pH dependence of the inactivation shows that two ionizable groups on the enzyme are involved in the inactivation. On the other hand, the steady-state kinetic behavior of the enzyme is welldescribed by ionization of a single group with a p K of 6.0 in the free enzyme. The basic form of the group is required for catalysis; protonation of the group decreases both V max and the apparent affinity for substrate. Conversely, binding of substrate decreases the p K of this group to about 5. l-Dihydroorotic acid is shown to be a competitive inhibitor of dihydropyrimidine amidohydrolase. Binding of l-dihydroorotic acid increases the p K of the ionizable group to 6.5. The agreement between the p K in the enzyme- l-dihydroorotic acid complex and the higher p K observed in the pH dependence of inactivation by chelators suggests that the same group is involved in the binding of substrate, l-dihydroorotic acid, and chelators. The different effects of substrate and l-dihydroorotic acid on the pK suggest that the binding modes of these two ligands may be different and suggest a structural basis for the mutally exclusive substrate specificities of dihydropyrimidine amidohydrolase and dihydroorotase.

Forskolin sensitizes human platelet adenylate cyclase to modulation of substrate (MgATP) affinity by hormones.

Stimulation of human platelet adenylate cyclase by the diterpene forskolin is associated with a decrease in the apparent substrate (MgATP) affinity of the enzyme. Addition of the stimulatory hormone prostaglandin E1 not only further increased the Vmax. of the forskolin-stimulated platelet adenylate cyclase but also caused a further increase in the Km value for MgATP, by up to 20-fold compared with basal conditions. On the other hand, the inhibitory hormone adrenaline decreased not only the Vmax. but also the Km value of the platelet adenylate cyclase stimulated by forskolin, with or without prostaglandin E1 present. The data indicate that forskolin sensitizes human platelet adenylate cyclase to modulation of substrate (MgATP) affinity by hormones, but there is no such effect in the absence of the diterpene.

Uptake of intact nucleoside monophosphates by Bdellovibrio bacteriovorus 109J.

The degraded nucleic acids and ribosomes of its prey cell provide Bdellovibrio bacteriovorus 109J with a source of ribonucleoside monophosphates and deoxyribonucleoside monophosphates for biosynthesis and respiration. We demonstrate that bdellovibrios, in contrast to almost all other bacteria, take up these nucleoside monophosphates into the cell in an intact, phosphorylated form. In this way they are able to assimilate more effectively the cellular contents of their prey. Studies with UMP and dTMP demonstrate that they are transported and accumulated against a concentration gradient, achieving internal levels at least 10 times the external levels. Treatment of the bdellovibrios with azide or carbonyl cyanide m-chlorophenylhydrazone eliminates their ability to either transport or maintain accumulated UMP and suggests the presence of a freely reversible exchange mechanism. There are at least two separate classes of transport systems for nucleoside monophosphates, each exhibiting partial specificity for either ribonucleoside monophosphates or deoxyribonucleoside monophosphates. Kinetic analyses of UMP transport in different developmental stages of strain 109J indicate that each stage expresses a single, saturable uptake system with a distinct apparent substrate affinity constant (Kt) of 104 microM in attack phase cells and 35 microM in prematurely released growth phase filaments. The capacity for transport of UMP by the growth phase filaments was 2.4 times that of the attack phase cells. These data, in addition to the apparent lack of environmental control of UMP transport capacity in attack phase cells, suggest that there are two transport systems for UMP in bdellovibrios and that the high-affinity, high-capacity growth phase system is developmentally regulated.

1-Naphthol conjugation in isolated cells from liver, jejunum, ileum, colon and kidney of the guinea pig

Cells from liver, jejunum, ileum, colon and kidney of the guinea pig were freshly prepared by standard isolation procedures. Cells were incubated in the presence of several concentrations of 1( 14C)-naphthol, and formation of 1-naphthol glucuronide and 1-naphthol sulphate was assessed at various times by thin layer chromatography. Cells from all five tissues conjugated 1-naphthol. The velocity of conjugation was fastest in jejunals cells (2.9 nmole/mg prot × min) followed by cells from liver, ileum, colon and kidney (0.2 nmole/mg prot × min). The apparent K m -values for glucuronidation ranged from 17 μM to 32 μM, and those for sulphation from 15 μM to 35 μM. Each tissue had a specific conjugation pattern. The two extremes were kidney cells, which had a glucuronidation/sulphation ratio in excess of 10, and colon cells which had a ratio of 0.38. The data suggest that these tissues possess different levels of 1-naphthol-conjugating enzymes, which resemble with regard to their apparent substrate affinities.

Reductive methylation of the two 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate-binding lysine residues of band 3, the human erythrocyte anion transport protein.

The bifunctional anion transport inhibitor 4,4'p-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) can react covalently with both chymotryptic peptides of band 3, the human erythrocyte anion transport protein. The functional roles of the two sites of covalent H2DIDS attachment have been investigated using reductive methylation with formaldehyde and borohydride, which is a substrate for band 3. Reductive methylation of outward facing lysing residues of intact cells can block the covalent H2DIDS reaction with both the 60,000- and the 38,000-dalton chymotryptic peptides (CH60 and CH38, respectively). The methylation does not interfere with reversible H2DIDS binding. Methylation of all copies of and 3 at both sites of covalent H2DIDS attachment inhibits Cl-Cl exchange by 75%. The same treatment does not detectably alter the apparent affinity for extracellular substrate (Br). The extent of transport inhibition by reductive methylation correlates very well with the extent of blockage of the Amino acid analysis indicates that the H2DIDS-binding lysine on CH38 rather than some coincidentally modified lysine is responsible for the inhibition. This lysine is therefore likely to be closely associated with the anion transport pathway.

Severe-glucose-6-phosphate dehydrogenase (G6PD) deficiency associated with chronic hemolytic anemia, granulocyte dysfunction, and increased susceptibility to infections: description of a new molecular variant (G6PD Barcelona)

Molecular, kinetic, and functional studies were carried out on erythrocytes and leukocytes in a Spanish male with G6PD deficiency, congenital nonspherocytic hemolytic anemia (CNSHA), and increased susceptibility to infections. G6PD activity was absent in patient's red cells and was about 2% of normal in leukocytes. Molecular studies using standard methods (WHO, 1967) showed G6PD in the patient to have a slightly fast electrophoretic mobility at pH 8.0 with otherwise normal properties (heat stability at 46 degrees C, apparent affinity for substrates, optimum pH, and utilization of substrate analogues). Other tests showed the patient's granulocytes to engulf latex particles normally, but to have impaired reduction of nitroblue tetrazolium and ferricytochrome-c as well as reduced iodination. Chemotaxis and random migration of the patient's granulocytes were normal as were myeloperoxidase, leukocyte alkaline phosphatase (LAP), and ultrastructural features. The molecular characteristics of G6PD in the patient differed from those of all previously reported variants associated with CNSHA, so the present variant was provisionally called G6PD Barcelona to distinguish it from other G6PD variants previously described. Possible mechanisms for the severe deficiency of G6PD in erythrocytes and granulocytes was investigated by studies on the immunologic specific activity of the mutant enzyme.