Clinical phenotype of Gaucher disease in relation to properties of mutant glucocerebrosidase in cultured fibroblasts.

We have investigated several parameters of glucocerebrosidase in cultured skin fibroblasts from patients with various clinical phenotypes of Gaucher disease. In this study no strict correlation was found between the clinical manifestations of Gaucher disease and the parameters investigated in fibroblasts. These parameters included the specific activity of the enzyme in extracts towards natural lipid and artificial substrate in the presence of different activators; the enzymic activity per unit of glucocerebrosidase protein; the rate of synthesis of the enzyme and its stability; and the post-translational processing of the enzyme. In addition, the activity in situ of glucocerebrosidase in fibroblasts was investigated using a novel method by analysis of the catabolism of NBD-glucosylceramide in cells that were loaded with bovine serum albumin-lipid complexes. Again, no complete correlation with the clinical phenotype of patients was detectable. Glucocerebrosidase in fibroblasts from most non-neuronopathic (type 1) Gaucher disease patients differs in some aspects from enzyme in cells from patients with neurological forms (types 2 and 3). The stimulation by activator protein and phospholipid is clearly more pronounced in type 1 than in types 2 and 3; the enzymic activity per unit of glucocerebrosidase protein in type 1 is severely reduced in the presence of taurocholate and the amount of glucocerebrosidase appears (near) normal in contrast to the situation in types 2 and 3 Gaucher fibroblasts. However, this distinction was not always consistent; glucocerebrosidase in fibroblasts from some type 1 Gaucher patients, particularly some South African cases, was comparable in properties to enzyme in type 2 and 3 patients.     

Gaucher disease. III. Substrate specificity of glucocerebrosidase and the use of nonlabeled natural substrates for the investigation of patients.

A reproducible and convenient method for assaying glucocerebrosidase activity using the natural substrates has been developed. From the insoluble pellet fraction of cultured skin fibroblast homogenates, released glucose was measured enzymically using hexokinase coupled with the glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP) system. Optimal enzyme assay conditions required both Triton X-100 and sodium taurocholate, pH 4.8. Glucocerebrosidase activities from three patients with type 1 Gaucher disease were 17.5%, 15.8%, and 11.2% of normal (normal = 198 +/- 14 nmol/hr per mg protein, n = 3). The first patient had normal beta-glucosidase activity with the artificial fluorogenic umbelliferone substrate. Interference with the accuracy of the glucose-dependent assay system by either glycolytic or gluconeogenic enzyme activites was not detected under these experimental conditions, and when substrates with long fatty-acid chain lengths (C = 22) were used, markedly decreased glucocerebrosidase activity occurred in both normal individuals and patients. The apparent Km's for the natural substrates were 0.56 +/- 0.05 mM for controls and 2.2-3.3 mM for Gaucher fibroblasts. These data further support the hypothesis that a structurally altered and catalytically deficient enzyme is synthesized in patients with type 1 Gaucher disease and illustrate the value of the natural substrate in investigating patients.     

Studies on the turnover of glucocerebrosidase in cultured rat peritoneal macrophages and normal human fibroblasts

The kinetics of glucocerebrosidase synthesis and degradation in rat peritoneal macrophages and in human fibroblasts have been studied using conduritol B epoxide (CBE), an irreversible and specific inhibitor of mammalian glucocerebrosidase. In cultured fibroblasts, higher concentrations of CBE and/or longer times were required for inhibition of glucocerebrosidase than were necessary for inhibition of the macrophage enzyme. However, inhibition of activity in cell extracts from both cell types showed identical time and concentration dependence. After the removal of CBE from cultures, enzyme activity returned to normal with a half-time of 48 h for macrophages and 40 h for fibroblasts. The reappearance of enzyme activity was prevented by an inhibitor of protein synthesis. Both the rate of synthesis and degradation of glucocerebrosidase enzyme protein were independent of the presence of CBE. The calculated rate of degradation of glucocerebrosidase was confirmed using metabolically labelled enzyme in cell cultures. The rate of synthesis for macrophages is 1.8 ng enzyme h-1 mg cell protein-1 and the rate of degradation is 1.4% h-1 (0.014 h-1). These values were 2.0 ng h-1 mg-1 and 0.018 h-1 for fibroblasts.     

Purification and the effect of peptide N-glycosidase F on lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts.

Glucocerebrosidase was purified from human cultured dermal fibroblasts more than 2200-fold to apparent homogeneity using high performance Alkyl-Superose HR 5/5 hydrophobic interaction and Bio-Sil TSK-250 gel permeation column chromatography. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis and protein staining of the catalytically active and concentrated enzyme fractions from the gel permeation columns revealed the presence of one band of Mr 64,000. The glucocerebrosidase preparation purified to homogeneity was digested with peptide N-glycosidase F that cleaves N-linked oligosaccharide structures from glycoproteins. The molecular weight of glucocerebrosidase after digestion with peptide N-glycosidase F was reduced to Mr 57,000, suggesting that the mature enzyme is a glycoprotein and that N-linked oligosaccharide constitutes a minimum of about 10% of the total molecular weight of the polypeptide. These findings are compatible with the hypothesis that glucocerebrosidase was initially synthesized as a precursor polypeptide which was subsequently glycosylated to become the mature enzyme.     

Purification and characterization of bovine brain glucocerebrosidase

Glucocerebrosidase was isolated from bovine brain by cholate extraction, ammonium sulfate fractionation, acid precipitation at pH 5.35, and hydrophobic chromatography. The purification is about 2400-fold with a specific activity of about 286,000 nmole/hr/mg protein. Molecular weight as determined by chromatography on Bio-Gel P-200 was 138,000. On SDS-polyacrylamide gel electrophoresis the enzyme protein resolved into two bands with apparent molecular weights of 63,000 and 56,000. These bands are cross-reactive to monospecific polyclonal antibody to homogeneous human placental glucocerebrosidase. The enzyme was found to be a complex glycoprotein based on its lectin binding specificity. Brain enzyme was found to be similar to placental glucocerebrosidase in its pH optima, heat stability at 52 degrees C, and substrate affinity. Enzyme kinetics were measured in the presence of conduritol-beta-epoxide, an irreversible inhibitor, and gluconolactone, a competitive inhibitor.     

The human glucocerebrosidase gene has two functional ATG initiator codons.

Gaucher disease is due to a deficiency in the activity of the enzyme glucocerebrosidase. Glucocerebrosidase is a lysosomal enzyme that presumably requires a signal peptide for transport across the membrane of the rough endoplasmic reticulum and glycosylation for transport into lysosomes. Human glucocerebrosidase cDNA contains two potential ATG start codons in its long open reading frame. The signal peptides that are initiated from each ATG are quite different in their hydrophobicity. We demonstrate that either ATG can function independently to produce active glucocerebrosidase enzyme in cultured fibroblasts. The glucocerebrosidase activity produced from translation products initiated at either ATG is found predominantly in the lysosomes.     

Identity of `acid'' β-glucosidase and glucocerebrosidase in human spleen

1. Glucocerebrosidase, in association with a membrane-bound ;acid' beta-glucosidase, was separated from a soluble ;neutral' beta-glucosidase that had no activity towards glucocerebroside as substrate. 2. Glucocerebrosidase, as well as ;acid' beta-glucosidase activity depended upon the association of factor P (a heat-stable, soluble, acidic glycoprotein) with factor C (a heat-labile membrane-bound protein). 3. Factor C was solubilized under certain conditions. 4. Solubilized factor C, as well as membrane-bound factor C, could be alternatively stimulated by sodium taurocholate to give both glucocerebrosidase and ;acid' beta-glucosidase activities. 5. Membrane-bound factor C reacted optimally with factor P whereas solubilized factor C was preferentially stimulated by taurocholate. 6. Factor P-dependent glucocerebrosidase activity differed in kinetic properties from the taurocholate-stimulated enzyme activity. 7. The results are discussed in the light of (a) identity of glucocerebrosidase and ;acid' beta-glucosidase, (b) application in clinical diagnosis, (c) physiological significance of the enzyme system, and (d) polygenic inheritance in adult Gaucher's disease.     

Alkaline phosphatase in hematopoietic tumor cell lines of the mouse: high activity in cells of the B lymphoid lineage

Alkaline phosphatase (EC 3.1.3.1) was assayed in a large number of cultured mouse tumor cell line using p-nitrophenylphosphate as the substrate. Of 19 lines of the B lymphoid lineage, including Abelson pre-B, B lymphoma, and plasma cell tumor lines, all but 1 had substantial activity averaging 407 nmol/min/mg protein (with a range from 5 to 900). Nine T lymphoid and 9 nonlymphoid hematopoietic lines examined had low activity of 0.7 to 4.2 nmol/min/mg protein. The enzyme was markedly enriched in plasma membrane preparations from the B lymphoid cells, but not in those from most T lymphoma cells. The activity of another plasma-membrane-bound enzyme, gamma-glutamyl transferase, did not vary systematically with the type of cell line but was exceptionally high in 1 T lymphoma line. Investigation of pH dependence and susceptibility to inhibition by L-phenylalanine and L-homoarginine indicated similarity of the alkaline phosphatase from B cell lines to the enzyme recoverable from normal mouse kidney, placenta, bone marrow, and lymphoid organs. The enzyme seems to provide a useful marker for tumor lines of the B lymphoid lineage and for their plasma membranes.     

Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography

Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase

The nature and function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase, have been investigated in cultured human skin fibroblasts. Glucocerebrosidase is synthesised as a 62.5-kDa precursor with high-mannose-type oligosaccharide chains and an apparent native isoelectric point of 6.0-7.0. Subsequent processing of the oligosaccharide moieties to sialylated complex-type structures results in formation of 65-68-kDa forms of the enzyme with apparent native isoelectric points of 4.3-5.0. These forms are transported to lysosomes and subsequently modified by the sequential action of lysosomal exoglycosidases, finally resulting in a 59-kDa form with an isoelectric point near neutrality. The existence of oligosaccharide modification of the enzyme in the lysosomes is illustrated by the accumulation of different intermediate forms of glucocerebrosidase in mutant cell lines deficient in lysosomal exoglycosidases. The enzyme does not undergo proteolytic modification during maturation. The possible physiological relevance of the oligosaccharide modification of glucocerebrosidase in the lysosomes was investigated by studying the properties of the enzyme in fibroblasts deficient in lysosomal exoglycosidases, and also the properties of homogeneous pure glucocerebrosidase from placenta, modified in the oligosaccharide moieties by digestion in vitro with glycosidases. Modification of the oligosaccharide moieties of glucocerebrosidase had no significant effect on the catalytic activity of the enzyme as measured with either artificial or natural substrates in the presence of artificial or natural activators. There was also no effect of modification of the oligosaccharide chains on the intracellular stability of the enzyme or on its apparent hydrophobicity. We conclude that oligosaccharide modification of glucocerebrosidase in the lysosomes simply reflects further maturation of the enzyme in the lysosome and is of no importance to its function.     

Improved estimation of DNA fragment lengths from Agarose gels

A simple, sensitive assay for prolylcarboxypeptidase (PCP) is described. It utilizes a radiolabeled substrate, benzyloxycarbonyl-l-prolyl-l-[³H]alanine, and the details of its synthesis are also reported here. The hydrolysis of the dipeptide substrate is linear with respect to time or protein concentration until 10% of the substrate has been cleaved. Kinetic analysis yielded a K_m of 4.7 mm. The assay can be used to measure PCP activity in small amounts of biological fluid, homogenized tissue or cultured cells. Measurements of PCP activity in various cultured human cells showed endothelial cells from umbilical veins to have the highest activity (1625 ± 151 nmol/mg/h) followed by endothelial cells from umbilical artery (1017 ± 46 nmol/mg/h), human foreskin fibroblasts (719 ± 39 nmol/mg/h), and pulmonary artery endothelial cells (352 nmol/mg/h).     

Lipoprotein lipase activity is stimulated by insulin and dexamethasone in cardiomyocytes from diabetic rats.

Type 1 diabetes mellitus reduces lipoprotein lipase (LPL) activity in the heart. The diabetic phenotype of decreased LPL activity in freshly isolated cardiomyocytes persisted after overnight culture (16 h). Total cellular LPL activity was 311+/-56 nmol oleate released x h(-1) x mg(-1) cell protein in diabetic cultured cardiomyocytes compared with 661+/-81 nmol oleate released x h(-1) x mg(-1) cell protein for control cultured cells. Diabetes also resulted in lower heparin-releasable (HR) LPL activity compared with control cells (111+/-25 vs. 432+/-63 nmol x h(-1) x mg(-1) cell protein). In kinetic experiments, the reduction in total cellular LPL and HR-LPL activities in cultured cells from diabetic hearts was due to a decrease in maximal velocity, with no change in apparent Km for substrate (triolein). LPL activity in primary cultures of cardiomyocytes from control rats is stimulated by the combination of insulin (Ins) and dexamethasone (Dex). Overnight treatment of cultured cardiomyocytes from diabetic rats with Ins+Dex elicited an 84% increase in cellular LPL activity (to 572+/-65 nmol x h(-1) x mg(-1) cell protein) and a 194% increase in HR-LPL activity (to 326+/-46 nmol x h(-1) x mg(-1) cell protein). This stimulation occurred at subnanomolar concentrations of the hormones, but neither hormone was effective alone. The amount of immunoreactive LPL protein mass in cultured cardiomyocytes from diabetic hearts was unchanged by Ins+Dex treatment. Addition of oleic acid (60 microM) to the overnight culture medium inhibited the already reduced HR-LPL activity in diabetic cultured cells by 73% (to 30+/-4 nmol x h(-1) x mg(-1) cell protein). The presence of oleic acid also reduced hormone-stimulated HR-LPL activity. Increasing the glucose concentration in the culture medium to 26 mM had no effect on total cellular LPL or HR-LPL activities.     

Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.     

Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells.

The membrane association of the lysosomal enzymes cathepsin D and glucocerebrosidase and its naturally occurring sphingolipid activating protein was studied in HepG2 cells. We differentially permeabilized cells with low concentrations of saponin, at which secretory proteins rinsed out completely, whereas integral membrane proteins were not released. All relevant intracellular compartments were shown to be permeabilized by saponin. Metabolic labeling showed that early precursors of cathepsin D, sphingolipid activating protein, and glucocerebrosidase were completely released from the cells, whereas more than 80% of the high molecular mass intermediates were retained by the cells. Treatment of permeabilized cells with 10 mM mannose 6-phosphate released only 50% of the cell-associated cathepsin D. Glucocerebrosidase remained membrane-associated, but cathepsin D and sphingolipid activating protein were released from the cells after proteolytic processing. Sphingolipid activating proteins and cathepsin D behaved similarly during biosynthesis and showed similar sensitivity to mannose 6-phosphate. The membrane association of the intermediate form of cathepsin D was independent of the presence of N-linked oligosaccharides. Subcellular fractionation on sucrose gradients showed that the lysosomal proteins became membrane-associated probably in the Golgi complex, and that both mannose 6-phosphate-dependent and mannose 6-phosphate-independent membrane association occur in the same compartments. We conclude that, in HepG2 cells, cathepsin D, sphingolipid activating protein, and glucocerebrosidase exhibit MPR-independent membrane association which is acquired in the same compartments beyond the rough endoplasmic reticulum.     

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture

Inorganic phosphate, amino acids and sugars are of obvious importance in lung metabolism. We investigated sodium-coupled transports with these organic and inorganic substrates in type II alveolar epithelial cells from adult rat after one day in culture. Alveolar type II cells actively transported inorganic phosphate and alanine, a neutral amino acid, by sodium-dependent processes. Cellular uptakes of phosphate and alanine were decreased by about 80% by external sodium substitution, inhibited by ouabain (30 and 41%, respectively) and displayed saturable kinetics. Two sodium-phosphate cotransport systems were characterized: a high-affinity one (apparent Km = 18 microM) with a Vmax of 13.5 nmol/mg protein per 10 min and a low-affinity one (apparent Km = 126 microM) with a Vmax of 22.5 nmol/mg protein per 10 min. Alanine transport had an apparent Km of 87.9 microM and a Vmax of 43.5 nmol/mg protein per 10 min. By contrast, cultured alveolar type II cells did not express sodium-dependent hexose transport. Increasing time in culture decreased Vmax values of the two phosphate transport systems on day 4 while sodium-dependent alanine uptake was unchanged. This study demonstrated the existence of sodium-dependent phosphate and amino acid transports in alveolar type II cells similar to those documented in other epithelial cell types. These sodium-coupled transports provide a potent mechanism for phosphate and amino acid absorption and are likely to play a role in substrate availability for cellular metabolism and in regulating the composition of the alveolar subphase. The decrease in phosphate uptake with time in culture is parallel to decrease in surfactant synthesis reported in cultured alveolar type II cells, suggesting that phosphate availability for surfactant synthesis may be accomplished by a sodium-dependent phosphate uptake.     

Synthesis of a fluorescent derivative of glucosyl ceramide for the sensitive determination of glucocerebrosidase activity

A fluorescent derivative of glucosyl ceramide was synthesized by covalently linking a fluorescent fatty acid, 12-[N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminododecanoic acid to the amino group of sphingosyl-1-O-beta-D-glucoside, glucosyl sphingosine. For hydrolysis by glucocerebrosidase, this substrate was dispersed in mixed micelles with Triton X-100 and sodium taurocholate or in unilamellar liposomes with phosphatidylcholine and the negatively charged lipid, dicetylphosphate. In either micellar or liposomal dispersions of the fluorescent substrate, reaction rates were linear with time and protein concentration, and saturation kinetics were observed. The rate of hydrolysis of this fluorescent substrate was equal to that obtained with radiolabeled glucosyl ceramide. The fluorescent glucosyl ceramide was used to determine glucocerebrosidase activity in extracts of human leukocytes, cultured skin fibroblasts, and various tissues as well as in partially purified splenic and placental glucocerebrosidase preparations. This fluorescent derivative of the natural substrate was not hydrolyzed by aryl beta-glucosidase(s), thereby facilitating the specific and reliable diagnosis of heterozygotes and homozygotes with Gaucher disease.     

Lysophosphatidylserine-dependent interaction between rat leukocytes and mast cells

The production of lysophosphatidylserine has been studied in a population of rat peritoneal cells; 67% polymorphonuclear and 33% mononuclear leukocytes. Pulse-chase experiments with L-[U-14C]serine reveal a net lysophosphatidylserine production of 0.33 nmol/mg protein in 2 h of incubation. The source of lysophosphatidylserine is probably the phosphatidylserine of cells damaged during the incubation, since plasma membrane fragments obtained from the leukocytes yield higher lysophosphatidylserine production (1.9 nmol/mg protein in 1 h of incubation). Both leukocytes and plasma membranes show phosphatidylserine splitting activity when tested with vesicles of this phospholipid. In the presence of albumin a fraction of produced lysophosphatidylserine is recovered in the incubation medium. Under these conditions efficient incorporation of lysoderivative into surrounding leukocytes and conversion to phosphatidylserine requires cell activation by tetradecanoylphorbol acetate. In agreement with radiochemical data it is found that a suspension of leukocytes elicits histamine release when rat peritoneal mast cells and nerve growth factor are subsequently added. This typical, lysophosphatidylserine-dependent mast cell response is retained when leukocyte plasma membranes substitute the whole cells. These results suggest that leukocyte lysis at sites of tissue injury results in the production of a sufficient amount of lysophosphatidylserine to reach and activate surrounding mast cells.     

Purification of human placental glucocerebrosidase using a two-step high-performance hydrophobic and gel permeation column chromatography method

Glucocerebrosidase was purified from human placenta approximately 10,600-fold to apparent homogeneity with an overall yield of 37% using cholate extraction, ammonium sulfate fractionation, butanol delipidation, and a two-step high-performance hydrophobic and gel permeation column chromatography method. A Phenyl-5PW (21.5 X 150 mm) column was used in the first step. Approximately one litre of delipidated and dialysed extract containing 3.7 X 10(6) units of enzyme activity from 1 kg of placental tissue was processed by the column at a flow rate of 5 ml/min. Glucocerebrosidase was eluted using a linear cholate gradient (2-3%). There was a 50-fold purification and 89% recovery. The run was completed in about 7 h. In the second step, the concentrated enzyme preparation from the phenyl column was run through two Bio-Sil TSK 250 gel permeation columns (21.5 X 600 mm) connected in series at a flow rate of 1.5 ml/min. A symmetrical peak of glucocerebrosidase activity (Ve = 253 ml) which had constant specific activity (47,000 units/h/mg protein) was noted. There was a 17-fold purification and 80% recovery in this run which was completed in 4 h. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and protein staining with silver compounds of the purified preparation revealed the presence of one band of Mr 68,000.     

An ectophosphatase activity in Candida parapsilosis influences the interaction of fungi with epithelial cells

This study describes the biochemical characterization of a phosphatase activity present on the cell surface of Candida parapsilosis, a common cause of candidemia. Intact yeasts hydrolyzed p-nitrophenylphosphate to p-nitrophenol at a rate of 24.30+/-2.63 nmol p-nitrophenol h(-1) 10(-7) cells. The cell wall distribution of the Ca. parapsilosis enzyme was demonstrated by transmission electron microscopy. The duration of incubation of the yeast cells with the substrate and cell density influenced enzyme activity linearly. Values of V(max) and apparent K(m) for p-nitrophenylphosphate hydrolysis were 26.80+/-1.13 nmol p-nitrophenol h(-1) 10(-7) cells and 0.47+/-0.05 mM p-nitrophenylphosphate, respectively. The ectophosphatase activity was strongly inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium orthovanadate, sodium molybdate, sodium fluoride, and inorganic phosphate, the final product of the reaction. Only the inhibition caused by sodium orthovanadate was irreversible. Different phophorylated amino acids were used as substrates for the Ca. parapsilosis ectoenzyme, and the highest rate of phosphate hydrolysis was achieved using phosphotyrosine. A direct relationship between ectophosphatase activity and adhesion to host cells was established. In these assays, irreversible inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells.     

Arginyl residue modification of the sarcoplasmic reticulum ATPase protein.

Glucocerebrosidase, a membrane bound enzyme, can be solubilized by acetone precipitation and the resultant soluble enzyme activity demonstrated in the presence of acidic phospholipid, e.g. phosphatidylserine. This is the first report of the detergent-free solubilization of glucocerebrosidase.