Apparent Substrate Affinity Research Articles

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.

Inhibition of adenylate cyclase activity in the corneal epithelium by anti-inflammatory steroids

Adenylate cyclase activity in bovine corneal epithelial or rat kidney particulate fractions was completely inhibited by clinically employed levels of anti-inflammatory steroids. Dose-response studies using several steroids showed that dexamethasone phosphate was the most potent enzyme inhibitor, followed respectively by prednisolone phosphate and hydrocortisone phosphate. Enzyme inhibition occurred regardless of the level or mechanism of adenylate cyclase activation, although agonist-stimulated activity was more sensitive to inhibition than fluoride-stimulated activity. Steroids appear to cause a direct, reversible and non-competitive inhibition of adenylate cyclase, affecting both the apparent substrate affinity and maximum velocity of the enzyme. Application of high levels of anti-inflammatory steroids may block the reported cyclic AMP-mediated effects of increased epithelial wound closure rates and stimulated epithelial basement membrane protein synthesis in a healing cornea.

Effects of aromatic retinoid on non-keratinizing (intestinal) epithelium: biochemical and morphological studies.

Doses of 3 mg/kg Ro 10-9359 (in arachis oil) were daily administered to adult female Wistar rats by gastric tube for a period of 10 days. Control animals received corresponding quantities of arachis oil only. The body weight of all rats was registered daily. Samples of jejunum and ileum were processed for quantitative histochemical analysis of neutral alpha-glucosidase kinetics and for three-dimensional evaluation of the mucosal architecture. In addition, mucosal scrapings were prepared from these intestinal segments, and the specific sucrase activity was determined. For each animal data were pooled and analyzed by Wilcoxon (Wn) test. Body weight and all registered parameters in the jejunum of treated animals remained unchanged as compared to the controls. In the ileum, however, we found under aromatic retinoid an increase of sucrase activity (P = 0.02) and of mucosal surface per unit serosal area (P less than 0.05). The hydrolytic activity of neutral alpha-glucosidase (Vmax) showed a clear trend to increase at both the villus base and apex, whereas the apparent substrate affinity (Km) remained unaltered. Our results show that, in closes of 3 mg/kg/day, aromatic retinoid induces (1) an increase in mucosal surface area, apparently due to hyperproliferation of the absorptive epithelium in ileum, which could facilitate its absorptive capacity and (2) an increase of specific sucrase activity, which could result in an enhanced carbohydrate assimilation. These findings indicate that Ro 10-9359 in addition to its effects on keratinizing epithelia exerts a distinct influence on the structure and function of the intestinal epithelium.

Properties of acid and neutral cholesterol ester hydrolases in rat and pigeon aortas

The activity of cholesterol ester hydrolase was measured in subcellular fractions from rat and pigeon aortas using a glycerol-dispersed cholesterol oleate substrate preparation. The specific activity of acid cholesterol ester hydrolase (assayed at pH 5) in adventitia tissue fractions was 40–50 fold greater than in media-intima fractions from rat aorta. Soluble and particulate subcellular fractions from rat aorta (media-intima) were observed to have cholesterol ester hydrolase activity with both an acid (pH 4.5–5) and a neutral (pH 7.5) pH optimum. A comparison of the subcellular distribution of acid cholesterol ester hydrolase with the lysosomal marker enzyme, N-acetylglucosaminidase, suggests that the acid hydrolase activity originated in aortic lysosomes; the neutral cholesterol ester hydrolase was predominantly soluble. Acid and neutral cholesterol ester hydrolases could also be distinguished on the basis of the effects of MgCl 2 and NaCl on hydrolase activity and on rates of thermal denaturation. Both acid and neutral hydrolases from rat aorta (media-intima) were inhibited by chloroquine (half-maximal at 2–4 mM), and both hydrolases were characterized as having the same apparent affinity for the glycerol-dispersed cholesterol oleate substrate. Acid and neutral cholesterol ester hydrolases were also observed in preparations from pigeon aortas. The specific activity for both acid and neutral hydrolases was higher in atherosclerosis-susceptible White Carneau pigeon aortas in comparison to Show Racer pigeon aortas.

Lactose transport in Escherichia coli: effect of transmembrane potential difference on apparent substrate affinity.

Lactose transport in Escherichia coli: effect of transmembrane potential difference on apparent substrate affinity.

Patterns of apparent co-operativity in a simple random non-equilibrium enzyme–substrate–modifier mechanism. Comparison with equilibrium allosteric models

It has often been claimed that random non-equilibrium mechanisms can result in apparent homotropic and heterotropic effects in steady-state kinetics of the kind more usually attributed to intersubunit allosteric interactions. However, it has never been shown whether any simple random mechanism could in fact give patterns of apparent interaction similar to those predicted by the well-known allosteric models. The patterns of apparent substrate co-operativity and affinity given by the steady-state of a standard simple random substrate-modifier mechanism in which catalytic velocity is proportional to substrate binding have been analysed mathematically and numerically. All patterns possible with this model are described. Some of them rather resemble those possible with standard allosteric models, in that there is a high-affinity and a low-affinity form at zero and infinite modifier concentrations (or vice versa) which show Michaelian behaviour, apparent co-operativity passing through a maximum or minimum at intermediate affinities. Unlike the allosteric models the family of curves is in principle not symmetrical. The random model can also give behaviour not possible with the standard allosteric models, such as higher substrate affinity at intermediate modifier concentrations than at either zero or infinite modifier, with concomitant negative apparent substrate co-operativity, or a single change of sign of apparent substrate co-operativity. The analysis uses recently discovered simplified forms of steady-state equations for random models.

Effects of starvation and development on mitochondrial acetoacetyl-coenzyme A thiolase of rat liver.

The activity of the putative ketogenic beta-oxoacyl-CoA thiolase from mitochondria of rat liver increases with starvation, during neonatal life, and after the injection of glucagon. These changes are associated with alteration in ketonaemia. The changes in activities of this species of thiolase are not associated with significant alterations in the apparent affinity (Km) for the ketogenic substrate, acetyl-CoA. These results support a role for thiolase in the regulation of ketogenesis.

Effects of FSH on Cyclic Nucleotide Accumulation in Testes of Rats of Various Ages

The accumulation of cyclic AMP in rat testis in response to FSH is much greater in immature rat compared to adult. Addition of 3-isobutyl-1-methylxanthine (MIX), a potent in vitro inhibitor of cyclic nucleotide phosphodiesterase activity, potentiates the effects of FSH but does not restore the cyclic AMP response of adult rat to that of immature rat testis. The apparent substrate affinities for cyclic AMP and cyclic GMP hydrolysis are similar in immature and mature testis. Developmental changes in cyclic AMP and cyclic GMP phosphodiesterase specific activities fail to account for the reduced responsiveness of adult testis to FSH. These results suggest a defect in synthetic rather than hydrolytic mechanisms in FSH-responsive cells.

Genetic regulation of plasma cholinesterase in man

Individual variation in response to succinylcholine has stimulated investigations on the variations and genetic control of serum cholinesterase in man. It has been found that the level of cholinesterase varies from essentially no detectable activity to exceedingly high levels due to a number of different genetic mutations. Qualitative variations in the esterase are also inherited. The most common variant of the latter type is the atypical (dibucaine resistant) cholinesterase which differs from the normal esterase in its lower apparent affinity for choline ester substrates and for a number of inhibitors. On the basis of evidence derived from kinetic experiments, both the anionic site and the esteratic site of the atypical esterase are modified. These changes may be due to a difference in the primary structure of the enzyme at one position which affects both sites of the active center of the enzyme. The modified kinetic properties of the atypical esterase are expressed in both the major component (C 4 ) and the minor components (C 1 , C 2 and C 3 ) of the enzyme which is present in serum in multiple molecular forms. Component C 4 (molecular weight of about 300,000) can be converted to component C 3 by treatment with urea, and the latter transformed to a C 1 -like component by sulfhydryl reagents. Both C 1 components (native and derived) have molecular weights of about 80,000 and are approximately one-fourth as large as C 4 . This information should be useful in further studies on the structure of variant forms of the esterase and how the structural alterations account for altered kinetic properties of the variant cholinesterases.

Regulation of ribose metabolism in Escherichia coli: II. Evidence for two ribose-5-phosphate isomerase activities

1. 1. There are two distinct ribose-5- P isomerase activities in wild type Escherichia coli X289. The activities are distinguishable by temperature sensitivity, apparent substrate affinity, and susceptibility to glucose-6- P inhibition. 2. 2. The heat-labile activity (the one with the greater affinity for ribose-5- P) is required for growth on ribose. The heat-stable activity does not appear to function in the formation of ribulose-5- P from ribose-5- P in vivo, presumably a combined result of its lower affinity for ribose-5- P and the maintenance of a low intracellular ribose-5- P concentration through product inhibition of the ribokinase. Glucose-6- P is a strong non-competitive inhibitor of the heat-labile ribose-5- P isomerase. 3. 3. Two mechanisms of ribose metabolism in this organism are proposed; one anabolic, the other amphibolic.