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Biomedical subjects

R H Glew

Publications and source records attributed to R H Glew.

At least 145 records · Page 8Linked to original sources
PubMed1986

Nosocomial transmission of delta hepatitis.

A previously asymptomatic carrier of hepatitis B virus receiving chronic hemodialysis developed acute delta hepatitis. The patient regularly received dialysis treatments on the same machine as a parenteral drug abuser with hepatitis B surface antigen (HBsAg)-positive chronic hepatitis whose serum was strongly positive for delta antibody. The drug abuser had a major bleeding episode that caused extensive environmental contamination 3 months before onset of illness in the index patient. No other patients receiving dialysis or staff members had evidence of delta infection. A surgeon previously infected with hepatitis B from the same parenteral drug abuser also had delta antibody. Testing for delta virus is indicated for both HBsAg-positive parenteral drug abusers and patients with hemophilia receiving chronic hemodialysis. All patients who are HBsAg- and delta-positive should receive dialysis separately from patients who are HBsAg-positive and delta-negative. Susceptible patients on dialysis and staff should receive hepatitis B vaccine to protect against both hepatitis B and delta virus infection.

Adult↗
PubMed1985

Comparison of N-acyl phosphatidylethanolamines with different N-acyl groups as activators of glucocerebrosidase in various forms of Gaucher's disease.

The acidic phospholipid requirement of the predominant particulate beta-glucosidase of mammalian spleen and liver was investigated using a series of N-acyl derivatives of dioleoyl phosphatidylethanolamine (PE). The PE, a neutral phospholipid, was converted to an acidic lipid, (N-acyl)-phosphatidylethanolamine (NAPE) by acylation of the amino group with different fatty acyl chains. Lysosomal beta-glucosidases from rat liver and spleens of controls and patients with various types of Gaucher's disease were solubilized and delipidated by extraction with sodium cholate and 1-butanol. All members of the NAPE series tested were effective activators of the delipidated rat liver beta-glucosidase, and the stimulatory power of the NAPE family increased with increasing chain length of the fatty acid substitution. In contrast, dioleoyl-PE had no effect on beta-glucosidase activity. A heat-stable factor (HSF) purified from the spleen of a patient with Gaucher's disease significantly increased the sensitivity of the rat liver beta-glucosidase to all of the NAPE derivatives. The maximum stimulation achieved in the presence of HSF was independent of N-acyl chain length. Compared to the potent activator, phosphatidylserine (PS), (N-acetyl)-PE and (N-linoleoyl)-PE were half as effective as activators of beta-glucosidase from control human spleen. PS stimulated the beta-glucosidase of type 1 nonneurologic Gaucher's disease, but none of the NAPE compounds activated it. Neither PS nor any of the (N-acyl)-PE compounds could activate a delipidated preparation of beta-glucosidase obtained from the spleen of a neurologic case. These results indicate that although the presence of a net negative charge on a phospholipid confers upon it an ability to reconstitute beta-glucosidase activity to the normal, nonmutant enzyme, it is insufficient to permit differentiation of the various types of Gaucher's disease.

Acylation↗
PubMed1985

Properties of an acid phosphatase from Legionella micdadei which blocks superoxide anion production by human neutrophils.

The high-speed supernatant (100,000 g, 1 h) obtained after centrifuging a suspension of Legionella micdadei that had been freeze-thawed and sonicated contained (i) considerable acid phosphatase activity when assayed using 4-methylumbelliferyl phosphate (MUP) as the substrate, and a factor that blocked superoxide anion production by human neutrophils stimulated with f-Met-Leu-Phe. Chromatography of the extract on a hydroxylapatite column resolved two acids phosphatases (designated ACP1 and ACP2). Subsequent chromatography of ACP2 on a Sephadex G-150 column revealed coincident elution of phosphatase activity and neutrophil blocking activity. When heated at 45 degrees C for various periods of time, the phosphatase activity of the acid phosphatase preparation was lost at the same rate as the ability of the preparation to block superoxide anion production by neutrophils. Furthermore, preincubation of neutrophils and acid phosphatase together in the presence of a heteropolymolybdate complex that inhibits the phosphatase eliminated the effect of the L. micdadei phosphatase on neutrophil superoxide anion production. ACP2 had the following properties: pH optimum, 6.0; Km for MUP, 3.8 mM; isoelectric point, 4.5; substrate specificity, MUP greater than ADP greater than phosphoenolpyruvate greater than phosphothreonine greater than phosphoserine greater than phosphotyrosine; molecular weight (estimated by sucrose density gradient centrifugation and gel filtration chromatography), 71,000-86,000. These results indicate that a cell-associated phosphatase may play a role in the virulence of L. micdadei.

Acid Phosphatase↗
PubMed1985

High-resolution proton nuclear magnetic resonance studies of the glucocerebrosidase activator protein from Gaucher spleen.

A heat-stable protein factor (HSF) obtained from the spleen of a patient with Gaucher's disease that activates glucocerebrosidase was studied by 600-MHz proton NMR spectroscopy. Assignments for a number of aromatic and aliphatic resonances were made on the basis of spin-decoupling, pH-titration, and resolution-enhancement experiments. The upfield ring current shifted aliphatic region and the downfield aromatic region were examined by nuclear Overhauser effect (NOE) methods using both pulsed Fourier-transform spectroscopy and correlation spectroscopy. It was found that a number of upfield-shifted methyl groups and certain methylene groups of specific aliphatic amino acid residues are in proximity relationships with several aromatic residues, forming a compact hydrophobic clustering site. Of special interest, tyrosine A, phenylalanine A, tryptophan B1, and tryptophan B2 were found to be located close to a cluster of aliphatic residues, indicating that the hydrophobic site of the HSF is conformationally rigid and its tertiary structure very compact. A two-dimensional structural model of the hydrophobic site of HSF is proposed.

Gaucher Disease↗
PubMed1985

Characterization of the activation of rat liver beta-glucosidase by sialosylgangliotetraosylceramide.

We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H2O and D2O revealed that the activation of beta-glucosidase by GM1 was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme.

Animals↗
PubMed1985

Galactosylsphingosine inhibition of the broad-specificity cytosolic beta-glucosidase of human liver.

Glucosylsphingosine is a potent inhibitor of lysosomal glucocerebrosidase and the broad-specificity, cytosolic beta-glucosidase of human liver. In the present study, it was demonstrated that the broad-specificity beta-glucosidase was also inhibited by galactosylsphingosine. The inhibition was observed when the enzyme was assayed for beta-glucosidase, beta-galactosidase, beta-xylosidase, and alpha-arabinosidase activities. Inhibition was of the mixed-type and the degree of inhibition depended on the substrate. For example, galactosylsphingosine was a more potent inhibitor of beta-glucosidase activity (I0.5 = 0.3 mM) than beta-xylosidase activity (I0.5 = 1.2 mM). In addition, the observation that the broad-specificity, cytosolic beta-glucosidase was inhibited by hydrophobic glycosphingolipids prompted the definition of a revised purification procedure which took advantage of hydrophobic affinity chromatography. This revised purification scheme employed Octyl-Sepharose and yielded the largest (68,000 Da) and most active (470,000 nmol h-1 mg protein-1) beta-glucosidase preparation yet described. The beta-glucosidase preparation contained 19% serine and 17% glycine, while 24% of the total amino acids were hydrophobic. The results of this study document the presence of a sphingolipid binding site on the broad-specificity beta-glucosidase. The implications of galactosylsphingosine inhibition of cytosolic beta-glucosidase and the possible role of the enzyme in glycosphingolipid metabolism are discussed.

Amino Acids↗
PubMed1985

Characterization of Leishmania donovani acid phosphatases.

A crude membrane fraction from promastigotes of Leishmania donovani grown in a liquid culture medium containing 20% fetal calf serum was prepared by freeze-thawing, centrifugation (200,000 X g, 30 min), and extraction with 2% (w/v) sodium cholate. After removal of the bile salt by chromatography on a Sephadex G-75 column, the solubilized membrane protein fraction, rich in acid phosphatase activity, was chromatographed on columns containing concanavalin A-Sepharose, QAE-Sephadex, and Sephadex G-150 and G-100. Three distinct acid phosphatases were resolved: the major phosphatase activity (70% of the total) was L-(+)-tartrate-resistant (designated ACP-P1) and corresponds to the acid phosphatase localized to the outer surface of the parasite's plasma membrane; the other two phosphatases (ACP-P2 and ACP-P3) account for the remaining 30% of the particulate acid phosphatase activity, and both of these enzymes are L-(+)-tartrate-sensitive. Using a combination of sucrose density gradient centrifugation, gel filtration chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was determined that ACP-P1 is a 128,000-dalton protein composed of two subunits of 65,000-68,000 daltons. ACP-P1 has an isoelectric point of 4.1, a pH optimum of 5.5, hydrolyzes fructose 1,6-diphosphate, but no other sugar phosphates and dephosphorylates phosphotyrosine, yeast mannan, and the phosphorylated form of rat liver pyruvate kinase. ACP-P2 (pI, 5.4) and ACP-P3 (pI, 7.1) with molecular masses of 132,000 and 108,000 daltons, respectively, are both tartrate-sensitive and are distinguished from each other on the basis of their sensitivity to inhibition by polyanionic molybdenum complexes. These two phosphatases also have their pH optima in the pH 5.0-6.0 range, but have a considerably broader substrate specificity than ACP-P1.

Acid Phosphatase↗
PubMed1985

Sucrose gradient analysis of phospholipid-activated beta-glucosidase in type 1 and type 2 Gaucher's disease.

Using sucrose density gradients, differences in delipidated lysosomal beta-glucosidase isolated from control spleen and spleen from patients with nonneurologic (type 1) and neurologic (type 2) Gaucher's disease have been examined. The three enzymes differ in sedimentation properties as well as in their responsiveness to activation by phosphatidylserine and heat-stable factor. The control beta-glucosidase sedimented as an apparent 45,000-Da species whose activity was dependent upon the inclusion of exogenous sodium taurodeoxycholate in the assay medium. Preincubation with a mixture of phosphatidylserine and heat-stable factor converted the control enzyme to a faster-sedimenting form which exhibited considerable activity in the absence of exogenous bile salt. Spleen beta-glucosidase from a patient with type 1 Gaucher's disease exhibited an apparent molecular weight of 154,000 on sucrose gradients. Like the control enzyme, the activity of this form was bile salt dependent. Upon preincubation with phosphatidylserine and heat-stable factor, beta-glucosidase from the type 1 case was also converted to a faster-sedimenting form which was more active in the absence of sodium taurodeoxycholate than in the presence of the bile salt. Spleen beta-glucosidase from the patient with type 2 Gaucher's disease sedimented as a broad peak of activity in the most dense regions of the sucrose gradients, appearing to be much larger than the beta-glucosidase from either the control or the type 1 Gaucher's disease patient. The activity of this large species was strongly dependent upon bile salt, and was not affected by preincubation of the enzyme with phosphatidylserine and heat-stable factor. Using the chaotropic salt, sodium thiocyanate (0.15 M), the spleen beta-glucosidase isolated from the type 1 Gaucher's disease case was converted to a slower-sedimenting species. The control enzyme sedimented slightly farther into the sucrose gradients upon treatment with the NaSCN. Thiocyanate treatment had no effect on the spleen beta-glucosidase isolated from the case of type 2 Gaucher's disease.

Aged↗
PubMed1985

Further studies on the activation of glucocerebrosidase by a heat-stable factor from Gaucher spleen.

Using Sephadex G-75 and DEAE-cellulose column chromatography, an 8270-Da glycopeptide (designated Fragment II) has been isolated from a cyanogen bromide-formic acid digest of a heat-stable factor from Gaucher spleen which activates a lipid-depleted preparation of lysosomal glucocerebrosidase from human liver. Fragment II contains all of the activity present in the native heat-stable factor. Compared with the parent factor, Fragment II contains four fewer cysteine and methionine residues and one less of each of the following: aspartic acid, threonine, serine, valine, isoleucine, and leucine. Nearly all of the monosaccharides present in the parent heat-stable factor can be accounted for in Fragment II, including three glucosamine, three mannose, one sialic acid, and one fucose. By itself, Fragment II has little or no stimulatory activity; its major effect is to markedly increase the sensitivity of glucocerebrosidase to activation by phosphatidylserine. A mixture of 1 microgram phosphatidylserine and 2 micrograms of the cyanogen bromide fragment activates the lipid-depleted preparation of glucocerebrosidase 50% more than 30 micrograms phosphatidylserine alone. Analysis of the Km and Vmax of glucocerebrosidase at various hydrogen ion concentrations revealed that the heat-stable factor and phosphatidylserine together dramatically increase the catalytic efficiency (Vmax/Km) of glucocerebrosidase while making apparent three ionizable groups that participate in the catalysis. Phosphatidylserine alone recruits two ionizable groups, but catalytic efficiency is lower than when heat-stable factor is also present. Heat-stable factor alone has no discernable effect on the ionization of functional groups on the enzyme or on catalytic efficiency. By sucrose density gradient ultracentrifugation, it was shown that preincubation of rat liver glucocerebrosidase with phosphatidylserine and heat-stable factor shifted the enzyme completely from a 56,600-Da form to a 188,100-Da form. The activity of the slower sedimenting form of glucocerebrosidase was totally dependent upon exogenous bile salt activator, whereas the faster sedimenting form exhibited the same activity in the presence or absence of sodium taurocholate. It appears that the heat-stable factor promotes the transfer of phosphatidylserine to glucocerebrosidase, which, in turn, results in an increase in both the catalytic efficiency and size of the enzyme.

Amino Acids↗
PubMed1985

Leishmania donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production.

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.

Acid Phosphatase↗
PubMed1985

alpha-1-Antitrypsin metabolism in the protein-deficient weanling rat.

Protein-deficient weanling rats fed on a 30 g casein/kg diet for 3 weeks lost albumin but maintained the level of serum alpha-1-antitrypsin, the most abundant protease inhibitor in blood. alpha-1-Antitrypsins from malnourished rats and control rats (given 250 g casein/kg diet) differed; the protease inhibitor from protein-deficient animals: (1) was more acidic, (2) appeared slightly larger (57 400 v. 56 000 daltons) on sodium dodecyl sulphate (SDS)-polyacrylamide gels, (3) had a more acidic Pi type and increased anodal mobility at pH 8.9, (4) bound more concanavalin-A and contained more carbohydrate, specifically two to three extra sialic acid residues. The amino sugar and neutral sugar contents of both preparations of alpha-1-antitrypsin were the same. Analysis of the products of cyanogen-bromide cleavage revealed that alpha-1-antitrypsin preparations from protein-deficient rats contain an extra glycopeptide that was not present in alpha-1-antitrypsin from control animals. In vivo studies showed that the increased sialic acid content of alpha-1-antitrypsin of protein-deficient rats did not alter the half-life of the molecule in the blood of control rats. However, the fractional catabolic rate of alpha-1-antitrypsin from either well-nourished or protein-deficient rats was significantly (P less than 0.01) lower in protein-deficient rats than in control rats (0.0247/h v. 0.0406/h). The decreased fractional catabolic rate could not be explained by changes in hepatic mannosyl-, galactosyl- or N-acetylhexosaminyl receptors since liver perfusion studies showed that bovine serum albumin, when covalently modified separately with each of these ligands, was extracted from the perfusion medium as rapidly or more rapidly by livers from malnourished animals. Perfused livers from protein-deficient rats secrete three times more alpha-1-antitrypsin than do livers from well-nourished animals. The decreased fractional catabolic rate and increased rate of biosynthesis and secretion of the glycoprotein by livers from protein-deficient animals may account for the maintenance of alpha-1-antitrypsin levels during protein malnutrition.

Albumins↗
PubMed1985

Effects of maternal protein deprivation on the nutritional status and neutrophil function of suckling neonatal rats.

An animal model of neonatal protein deprivation was developed to examine the effects of maternal malnutrition on growth and development and on the host defense system of the suckling offspring. Adult rats were fed either a protein-deficient (3% casein) or normal (25% casein) diet beginning one day after parturition. Offspring of the protein-deprived animals showed biochemical signs of nutritional imbalance such as changes in serum acid hydrolase levels as early as the second day of life; growth retardation and hypoproteinemia developed by day 4. When malnourished and control sucklings were infected at 12 days of age with Staphylococcus aureus, it was noted that protein deprivation did not influence neutrophil mobilization. However, malnourished animals responded to infection with larger perturbations in neutrophil counts than did the controls, were unable to control the infection, and ultimately showed neutrophil depletion. These studies suggest that protein deprivation affects the quantity and quality of milk and that the offspring of a protein-deficient animal are not only growth retarded but are also compromised in their ability to deal with infection.

Animal Population Groups↗
PubMed1985

Comparison of the Beckman Auto ICS and the Syva Autolab 6000 for determination of gentamicin levels in serum.

Two fully automated drug-monitoring systems, the Syva Autolab 6000 and the Beckman Auto ICS, were compared in terms of accuracy, precision, speed of operation, and cost effectiveness in the determination of gentamicin levels in serum. Within-run and between-run precision for both systems were acceptable (coefficient of variation, 2.0 to 6.9%), and patient sample comparisons resulted in an intermethod correlation coefficient of 0.96. When reference samples (prepared to contain 1.2 to 10 micrograms of gentamicin per ml) were assayed, the Syva Autolab 6000 obtained concentrations within 8% of expected values, whereas the Beckman Auto ICS reported values up to 17% higher than target values. In a time and cost comparison, reagent costs for the Beckman system were ca. 50% less than for the Syva Autolab 6000; the Syva system, however, determined patient results two to three times faster than the Beckman Auto ICS.

Costs and Cost Analysis↗
PubMed1985

Resistance of leishmanial phosphatases to inactivation by oxygen metabolites.

Leishmania donovani promastigotes produce large quantities of two distinct acid phosphatases; a tartrate-resistant enzyme is localized to the external surface of the plasma membrane, and a tartrate-sensitive enzyme is secreted into the growth medium. It was shown previously that preincubation of human neutrophils and macrophages with the tartrate-resistant phosphatase markedly reduced the ability of these host cells to produce superoxide anions in response to stimulation with the activator formyl-methionyl-leucyl-phenylalanine. The possibility that the cell surface acid phosphatase or the phosphatase that is secreted into the extracellular fluid might compromise other host cell functions, especially intracellular ones, depends on the ability of the enzyme to resist exposure to toxic oxygen metabolites (e.g., superoxide anion, hydrogen peroxide, hypochlorite) generated by phagocytic cells. In the present report, we show that both leishmanial acid phosphatases were relatively resistant to inactivation by oxygen metabolites. At pH 5.5, the activity of the tartrate-resistant phosphatase was reduced 50% by incubation for 1 h with each of the following: 30 mM O2-, 500 mM hydrogen peroxide, and 6 mM hypochlorite ion. These concentrations are many fold greater than the concentrations of these substances that are generated by stimulated polymorphonuclear phagocytes. The tartrate-sensitive acid phosphatase differed markedly from the tartrate-resistant phosphatase in that the former was essentially insensitive to even very high concentrations of superoxide anion and hydrogen peroxide. Furthermore, 50% inactivation of the tartrate-sensitive leishmanial phosphatase required exposure to 35 mM hypochlorite for 30 min. These results indicate that the catalytic potential of these two leishmanial acid phosphatases probably survives exposure to toxic oxygen metabolites generated by neutrophils and macrophages.

Acid Phosphatase↗
PubMed1984

Characterization of the phospholipid requirement of a rat liver beta-glucosidase.

The lipid requirement of membrane-bound rat liver beta-glucosidase was investigated using 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. The enzyme was solubilized and delipidated by sequential extraction of a crude lysosomal fraction from rat liver lysosomes with sodium cholate and ice-cold butan-1-ol. Neither saturated nor unsaturated phosphatidylcholine activated this enzyme. In contrast, acidic phospholipids like phosphatidylglycerol (PtdGro) and phosphatidylserine (PtdSer) were effective activators. For the PtdGro series, fatty acid composition was important, with the shorter chain or unsaturated fatty acid-containing PtdGro species being the best activators. Heat-stable factor (HSF) from Gaucher spleen by itself (1-2 micrograms) had no effect on enzyme activity. However, the same amount of HSF when combined with 10 micrograms of PtdSer markedly stimulated beta-glucosidase activity. In the presence of HSF, di-9-cis-octadecenoyl-PtdGro (1 microgram) or -PtdSer (5 micrograms) provided maximum protection of beta-glucosidase against heat (60 degrees C) inactivation. In the absence of phospholipids, HSF had no effect on the rate of inactivation of the enzyme by the suicide inhibitor conduritol B epoxide (t0.5, 12 +/- 0.5 min); the maximum rate of inactivation was achieved in the presence of a mixture of PtdGro (2.5-5 micrograms) and HSF (t0.5, 2.8 min). The combination of PtdSer (10 micrograms) and HSF (1.3 micrograms) lowered the Km for 4-methylumbelliferyl-beta-D-glucopyranoside from 24 to 2.7 mM. Inhibition of the enzyme by the glucocerebrosidase substrate analogues N-hexyl-O-glucosylsphingosine and glucosylsphingosine was influenced by the activator substances. The inclusion of PtdSer and HSF in the beta-glucosidase assay medium lowered the Ki of N-hexyl-O-glucosylsphingosine 20-fold. The same combination of activators decreased the I0.5 of the enzyme for glucosylsphingosine from 89.4 to 7.6 microM. A study of log (Vmax./Km) versus pH indicated that the PtdSer-HSF pair creates the active site of beta-glucosidase, making apparent three ionizable groups on the enzyme with pK values in the range 4.5-5.1.

Animals↗
PubMed1984

Leishmanial phosphatase blocks neutrophil O-2 production.

Leishmania donovani, the causative agent in kala-azar or visceral leishmaniasis, infects cells of the macrophage system. We show that a purified preparation of the tartrate-resistant acid phosphatase, isolated from the external surface of L. donovani promastigotes, inhibits superoxide anion production by human neutrophils. Preincubation of neutrophils for 15-30 min at 37 degrees C with 240 units (1 unit equals 1 nmol of 4-methylumbelliferyl phosphate cleaved per h) of the acid phosphatase decreases both the rate and extent of superoxide generation by 90% upon stimulation with the chemoattractant peptide fMet-Leu-Phe. The ability of the phosphatase to suppress superoxide anion production is abolished by heat inactivation of the enzyme or by incorporation of an acid phosphatase inhibitor in the preincubation medium, indicating that the effect is dependent on the catalytic activity of the enzyme. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.

Acid Phosphatase↗
PubMed1984

Calmodulin and parvalbumin: activators of human liver glucocerebrosidase.

Lysosomal glucocerebrosidase of human tissues is reversibly inactivated by extraction with sodium cholate and n-butanol. Enzyme activity can be restored in the glucocerebrosidase assay by the incorporation of small amounts of phosphatidylserine (1 micrograms/assay) and a heat-stable factor obtained from the spleen of patients with Gaucher's disease. In the present report, we show that two heat-stable, low-molecular-weight, acidic, calcium-binding proteins, namely calmodulin and parvalbumin, are relatively potent activators of human liver glucocerebrosidase. A third structurally related, calcium-binding protein, troponin-C, does not stimulate glucocerebrosidase significantly. Removal of calcium from these proteins by treatment with 5 mM ethylene glycol bis(beta-aminoethylether)-N,N'-tetraacetic acid greatly decreases the quantity of material needed to stimulate enzyme activity. Parvalbumin stimulation of glucocerebrosidase activity is dependent on the presence of phosphatidylserine whereas the ability of calmodulin to activate the enzyme is not dependent on the acidic phospholipid. In terms of the level of glucocerebrosidase activity they support and under optimal conditions, parvalbumin and calmodulin are about 50 and 30%, respectively, as effective as the heat-stable factor from Gaucher spleen. On the other hand, on a molar basis, it takes about 35 times more parvalbumin than calmodulin to achieve maximum stimulation of glucocerebrosidase activity.

Calmodulin↗