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S Gatt

Publications and source records attributed to S Gatt.

At least 73 records · Page 4Linked to original sources
PubMed1989

Correlation of the dispersion state of pyrene cerebroside sulfate and its uptake and degradation by cultured cells.

This study aimed at increasing the efficiency and shortening the time required for administering cerebroside sulfate to cultured cells. For this purpose several modes of dispersion of a fluorescent derivative of cerebroside sulfate (sulfatide), in which the natural fatty acid has been replaced by pyrenedodecanoic acid (P12), were incubated with the cells. This fluorescent derivative of cerebroside sulfate (P12-CS) was introduced into the growth medium of the cells using the three following modes of dispersion: (1) P12-CS was dissolved in dimethylsulfoxide and added to the medium, (2) it was precomplexed with serum albumin or (3) incorporated into small, unilamellar vesicles (SUV) of phosphatidylcholine. With each of these respective modes of dispersion, the P12-CS was incubated for periods up to 48 h with cultured lymphoblasts or fibroblasts. Uptake by the cells could be determined by recording directly the cell-associated fluorescence, using a suspension of washed intact cells. The cell lipids were subsequently extracted with mixtures of chloroform/methanol and their fluorescence recorded. When related to the incubation time, uptake of P12-CS by the cells increased continuously using each of the above dispersions. The appearance of fluorescence at 475 nm ('excimer') and the ratio of this to the monomolecular fluorescence at 378 nm ('E/M') could be used as a measure for the presence of the internalized P12-CS in aggregated or fully dispersed states. These values (i.e., E/M), recorded on the suspensions of intact cells were rather high using the aqueous dispersions, intermediate values were observed using the SUV and rather low E/M values (0.5 or less) were observed using the preformed albumin-(P12-CS) complexes. Increasing the mole ratio of albumin to P12-CS (i.e., from 1:2 to 2:1 m/m), decreased the quantity of sulfatide which was taken up by the cells but also further decreased the E/M ratio, suggesting a fully dispersed state of the pyrene lipid within the cell. This indicated that, using an optimal albumin to P12-CS ratio of 1-2 (or its equivalent values in fetal calf serum) permitted an influx of single molecules of P12-CS into the cells. After 48 h, about 50% of the fluorescence of skin fibroblasts was found in metabolic degradation products of P12-CS. The parallel value for fibroblasts derived from a patient with metachromatic leukodystrophy was only about 5%. Appearance of the excimeric emission of a dispersion of P12-CS in water permitted estimation of its critical micellar concentration as being 7.5 x 10(-7) M.

Cell Line↗
PubMed1989

Analysis of cellular heterogeneity in the response of human leukemic cells to photosensitization induced by pyrene-containing fatty acid.

Incubation of cells with 12-(1-pyrene) dodecanoic acid (P12) followed by irradiation with ultraviolet light at 366 nm (UVA) resulted in cytotoxicity. We compared the photosensitivity to UVA irradiation of various human myelo-monocytic leukemic cell lines, their intra- and inter-clonal variability and correlated their photosensitivity to P12-uptake and metabolism. The fluorescence properties of pyrene were utilized for flow cytometric analysis of cell distribution with respect to P12-uptake as well as for sorting subpopulations differing in their fluorescence. Spectrofluorometric analysis of the total cell-associated fluorescence and of the cellular lipids-associated fluorescence were also carried out. Considerable heterogeneity in P12-uptake and photosensitivity was found not only among cell lines, but also in the response of different clones and among the individual cells in specific clonal populations. Within a clone, photosensitivity was related to the amount of P12 taken up by the individual cells, while among different cell lines and their clones the photosensitivity was correlated with the proportion of cellular pyrene-linked phospholipids. The larger the fraction of pyrene-linked phospholipids within the cell--the more sensitive it was to UVA-irradiation. Photosensitivity could be affected by changing the proportion of cellular pyrene-linked phospholipids. Cells treated with cAMP showed an increase in total P12-uptake, but the proportion of pyrene-linked phospholipids was reduced, resulting in lower photosensitivity. These findings, demonstrating that by manipulating lipid metabolism photosensitivity can be modified, may prove useful in a clinical setting for selective photosensitization of malignant cells.

Cell Survival↗
PubMed1989

Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases.

Fluorescent pyrene-methyl lauryl ester (PMLes) was synthesized and used for the determination of cellular lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or cholesteryl ester storage diseases (both exhibiting a deficiency of the lysosomal acid lipase). The hydrolysis of PMLes by acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use PMLes as a fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (i.e. pyrene-methanol) was read in the lower aqueous-ethanolic phase, at 378 nm. PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or cholesteryl ester storage diseases. This demonstrates that the fluorescent PMLes is hydrolyzed by the lysosomal acid lipase and can be used as a very sensitive fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.

Cell Line↗
PubMed1988

Synthesis of pyrenesulfonylamido-sphingomyelin and its use as substrate for determining sphingomyelinase activity and diagnosing Niemann-Pick disease.

A new fluorescent derivative of sphingomyelin (PSA12-sphingomyelin) containing a pyrene-sulfonylamide residue was synthesized by covalently linking 12-((1-pyrenesulfonyl)amido)-dodecanoic acid (PSA12) to sphingosylphosphorylcholine. It was used as substrate for acidic and neutral human and murine sphingomyelinases permitting development of sensitive assays for these enzymatic activities. The product of the sphingomyelinase assay, PSA12-ceramide, could be detected in picomole quantities due to a fluorescence intensity which was 10-35-fold greater than that of other fluorescent ceramides (such as pyrene or nitrobenzoxadiazole derivatives). PSA12-sphingomyelin could be used in pure form or admixed with natural sphingomyelin; in the latter case, the enzyme hydrolyzed the fluorescent and non-fluorescent species at equal rates. Use of PSA12-sphingomyelin permitted determination of sphingomyelinase activity in cell extracts (eg human blood lymphocytes, lymphoid cell lines or cultured skin fibroblasts) as well as in hair follicles and urine. This new fluorescent derivative of sphingomyelin also permitted the detection of acid sphingomyelinase deficiency in cells derived from patients with Niemann-Pick disease.

Animals↗
PubMed1988

Spectrofluorometric measurements of the dispersion state of pyrenedodecanoic acid and its uptake by cultured cells and liposomes.

Pyrene dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene is covalently linked, showed a considerable increase in fluorescence when the probe was introduced into a hydrophobic environment. Also, when closely packed in an aggregate, an energy transfer between two adjacent molecules of pyrene occurred, resulting in a shift of the peak of the emission spectrum from 378 nm ('monomeric') to 475 nm ('excimeric'). These two respective properties were utilized for the following: (a) A spectrofluorometric measurement of the critical micellar concentration (CMC) of the pyrene fatty acid, defined as the concentration at which the 475 nm emission peak appeared as a consequence of the aggregation of P12 molecules in aqueous solution to form micelles; the CMC of P12 was found to be in the range of 1 to 2 microM. (b) The penetration of P12, from an aqueous solution or dispersion, into unilamellar phospholipid vesicles was determined by monitoring the increase of the fluorescence at 378 nm. The fluorescence increase was time-dependent and proportional to the respective concentrations of P12 or phospholipid vesicles. Substituting the neutral phosphatidylcholine with the negatively-charged phosphatidylserine vesicles resulted in a slower rate as well as lesser total uptake of P12. (c) The uptake of P12 by cells was accompanied by an increase in the monomeric fluorescence emission intensity. Using cells in suspension, this could be followed continuously in a spectrofluorometer equipped with a recorder. The uptake was found to be time-dependent and proportional to P12 concentration.

Animals↗
PubMed1988

Continuous spectrofluorometric measurements of uptake by cultured cells of 12-(1-pyrene)-dodecanoic acid from its complex with albumin.

Aqueous dispersions of 12-(1-pyrene)-dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene has been covalently linked, shows a considerable increase in fluorescence when the probe is introduced into a hydrophobic environment. This enables the uptake of P12 by liposomes and cells to be followed directly in a spectrofluorometer, without separating the cells from the P12-containing medium. In the present study, we show that complexing P12 to albumin produced a very high fluorescence emission intensity. This made direct measurements of the uptake by cells of albumin-bound P12 impossible. Such direct measurements could, however, be made using albumin which had been interacted with trinitrobenzenesulphonic acid (TNBS). The yellow trinitrophenyl (TNP) residues, which were thereby covalently linked to the albumin, quenched the fluorescence of pyrene in the TNP-albumin/P12 complex. Upon release of the P12 molecules from this complex and their subsequent uptake by cells, fluorescence increased. This technique was utilized for the continuous monitoring of the uptake of P12 by different cell types and cells at various stages of maturation.

Animals↗
PubMed1988

Relative fluorescence of normal and acid lipase-deficient cultured fibroblasts following administration of pyrene decanoic acid.

Skin fibroblasts, derived from normal individuals or patients with Wolman's disease (an autosomal recessive disorder due to acid lysosomal lipase deficiency) were incubated with the fluorescent fatty acid, pyrene-decanoic acid (P10). Measurements of the fluorescence intensities of the total lipid extracts indicated that equal quantities of P10 were incorporated into both cell types. The fluorescence emitted by the intact cells was subsequently recorded in a fluorescence microscope equipped with a microdetector unit, which permitted determination of the fluorescence emitted by the intact cell or by specific regions thereof. The fluorescence intensities emitted by the lipidotic cells exceeded those of their normal counterparts 2- and 5-fold when comparing the entire cells or the perinuclear region, respectively. The cells were then subjected to subcellular fractionation and an analysis of the fractions revealed that up to 85-90% of the fluorescence of the lysosome-mitochondrial pellet was derived from free pyrenedecanoic acid; the latter contributed only 15-18% to the fluorescence of the homogenate or the cytosol. There was no difference in the fluorescence of the lipid extracts from the respective fractions of the lipidotic or normal cells. However, the fluorescence emitted by the intact lysosome-mitochondrial fraction of the lipidotic cells exceeded that of its normal counterpart 2.5-fold. These data suggest that the increased fluorescence intensity of the intact lipidotic cells resulted from a higher quantum yield of free P10 molecules solubilized in the hydrophobic environment of their neutral lipid-containing storage granules.

Cell Compartmentation↗
PubMed1988

Pyrene dodecanoic acid coenzyme A ester: peroxisomal oxidation and chain shortening.

Pyrenedodecanoyl-CoA was beta-oxidized by isolated rat liver peroxisomes at a rate which was about 50% of that observed with palmitoyl-CoA. Measurement of the quantity of NADH formed from a limiting amount of pyrenedodecanoyl-CoA suggested that it was subjected to two to three cycles of beta-oxidation. Pyrenedodecanoyl-CoA was a very poor substrate for carnitine palmitoyltransferase, exhibiting less than 1% of the rate obtained with palmitoyl-CoA; it also was a strong inhibitor of this enzyme. With rat liver microsomal alpha-glycerophosphate acyltransferase the rate of reaction with pyrenedodecanoyl-CoA was only 3-4% of that observed with palmitoyl-CoA.

Acyl Coenzyme A↗
PubMed1988

Flow cytofluorometric analysis of the uptake of the fluorescent fatty acid pyrene-dodecanoic acid by human peripheral blood cells.

The fluorescence activated cell sorter (FACS) was used for measuring the uptake of the fluorescent fatty acid derivative 12-(1-pyrene) dodecanoic acid (P12) by human peripheral blood cells. The results indicate that blood cells differ widely in their ability to take up P12, with polymorphonuclear cells showing the greatest uptake, followed by lymphocytes, platelets, and RBCs. These differences in P12 uptake provide a potential additional parameter for differential cell counting. Using the ability of the FACS to "gate out" nonrelevant cells, it was possible to measure the rate of P12 uptake by each respective cell type even when admixed with other cells. Thus elaborate physical separation procedures could be avoided, and contaminating cells did not influence the results. Differences in P12 uptake were also utilized to separate blood cells into pure subpopulations of specific cell types.

Blood Cells↗
PubMed1988

Human acid beta-glucosidase: inhibition studies using glucose analogues and pH variation to characterize the normal and Gaucher disease glycon binding sites.

Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid beta-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., Kis much less than Kii) and had low apparent affinity for the enzyme (Kisapp = 20-500 mmol/l). beta-Glucose-1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-beta-glucoside hydrolysis in an uncompetitive pattern (Kiapp = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-beta-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5-imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with Kisapp(beta-glucose)/Kisapp approximately equal to 10(3)-10(5); an amine at C-2 did not alter Kisapp compared to beta-glucose. The variation of Kisapp with pH for the 5-imino- and 1-deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH2) of the diprotic (Vmaxapp and Vmaxapp/Kmapp) normal enzyme (pK1 = 4.7; pK2 = 6.7) with pH-independent Kisapp values of 2.9-9.0 mumol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that Kisapp(AJGD)/Kisapp(normal) = 4-17 for dNM and 1-deoxy-1-amino-beta-glucose; this ratio was approximately equal to 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid beta-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of beta-glucose and a residue with pKa = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid beta-glucosidase.

Adult↗
PubMed1987

Enhancement of fluorescence of pyrene-containing lipids by polar media, detergents and phospholipids.

The fluorescence intensities of a medium-chain fatty acid and of several amphiphilic lipids, each containing pyrene in covalent linkage, were enhanced considerably by: 1) Dissolving in mixtures of a polar solvent (e.g. methanol, ethanol, tetrahydrofuran or dimethylsulfoxide) and water; for each individual compound, a certain ratio of solvent to water provided maximal fluorescence intensity. 2) Incorporating into micelles of reduced Triton X-100; an excess of detergent was used so that, statistically, only one molecule of lipid resided in one micelle of the Triton X-100. 3) Incorporating into liposomes of egg phosphatidylcholine; maximal fluorescence was observed using a large excess of phosphatidylcholine. When related to the fluorescence intensities in chloroform/methanol (2:1, by vol.) or water, the enhancement of fluorescence in the above three systems was about 2-6-fold or up to 60-fold, respectively.

Ceramides↗
PubMed1987

Uptake and intracellular degradation of fluorescent sphingomyelin by fibroblasts from normal individuals and a patient with Niemann-Pick disease.

Sphingomyelin, labelled with a fluorescent probe, pyrene, in the fatty acyl residue was associated with fetal calf serum; approx. 80% of the sphingomyelin was found in the low- and high-density lipoproteins. This was added to the growth medium of cultured human skin fibroblasts from normal individuals and a patient with Niemann-Pick disease type A, devoid of acid sphingomyelinase activity. The fluorescent sphingomyelin was taken up by both cell types, but only the former degraded it to produce fluorescent ceramide. Differences between normal and Niemann-Pick cells in sphingomyelin content or ceramide production were observed after several hours uptake. A more pronounced difference was noted when cells were incubated for 1 day with fluorescent sphingomyelin and then for two to three days in medium devoid of this compound. Under these conditions, the fluorescence intensity of the Niemann-Pick cells remained practically constant while that of their normal counterparts was almost completely eliminated from the cells. Comparison of fluorescence intensities of these two cell types could be made directly on aqueous suspensions of whole cells or, alternatively, on their lipid extracts. For evaluation of the degradation of fluorescent sphingomyelin to ceramide within the cells, several procedures were developed for the rapid isolation of the latter compound from the total lipid extract. The results suggest that when associated with the constituents of the fetal calf serum, sphingomyelin is taken up by the cells and transported into the lysosomal compartment where it is degraded to ceramide. Use of the fluorescent derivative of sphingomyelin provided a simple and rapid procedure for following the uptake by and degradation within the cultured cells. It also permitted the establishment of differences in the rates of degradation of the fluorescent sphingomyelin by cells with a normal metabolism and others lacking sphingomyelinase (i.e., Niemann-Pick disease type A cells).

Biological Transport, Active↗
PubMed1987

Use of the fluorescence activated cell sorter for studying uptake of fluorescent fatty acids into cultured cells.

A procedure is described for the continuous monitoring and recording of fatty acid transport into cultured cells. Uptake of the fluorescent fatty acid derivative, 12-(1-pyrene)dodecanoic acid [P12] by a suspension of human promyelocytic leukemia (HL-60) cells was analyzed and recorded by the Fluorescence Activated Cell Sorter (FACS). A major advantage of the FACS is its ability for measuring the fluorescence of the cells only, without interference by that of the fatty acids dissolved or dispersed in the medium or complexed with serum albumin. The time-dependent fluorescence increase due to uptake of the acid by the cells could thus be monitored and recorded directly without a need for sedimenting, washing and extracting the cellular lipids. This procedure provided an accurate measurement of the kinetics of uptake of the fatty acid into the cells and permitted study of the effect of various parameters such as temperature, glucose, albumin or serum. The results indicated a biphasic uptake of P12 into the cells. The first, a rapid, energy-independent phase, lasted 3-4 min. This was followed by a slower uptake which was directly related to the metabolic utilization of the acid in the cells. This second phase represents the overall process of translocation across the cell membrane, activation to acyl coenzyme A and incorporation into the cellular neutral lipids and phosphoglycerides. When compared to HL-60, murine erythroleukemia (MEL) cells took up considerably lesser quantities of P12. This was utilized for setting up a model system, in which mixtures of these two respective cell types could be identified and separated from each other on the basis of their relative rates of uptake of the fluorescent fatty acid.

Animals↗
PubMed1987

Clonal analysis of the response of human promyelocytic leukemia (HL-60) cells to photosensitization induced by a pyrene-containing fatty acid.

Incubation of cells with 12-(1-pyrene) dodecanoic acid (P12), a fatty acid to which a pyrene nucleus has been covalently linked, followed by irradiation with long-wave ultra-violet light (LUV) at 366 nm, resulted in cytotoxicity. Syntheses of macromolecules was significantly decreased after 30 min, while an accumulation of trypan-blue positive, non-viable cells was observed several hours following irradiation. Cloning of the irradiated cells in semi-solid medium showed an exponential dose-response survival curve. Above a threshold dose colony number decreased, although the rate of clonal development and the final size were not affected. The sensitivity of detecting rare surviving cells could be increased by incubating the irradiated cells for several hours in liquid culture followed by concentrating intact cells by gradient sedimentation. Using this procedure, one surviving clonogenic cell could be detected in 10(7) irradiated cells/dish. More than 10 min of irradiation at 773 microV/cm was required to photosensitize the population below detection by this method. The possibility was considered that colonies derived from cells surviving sub-maximal LUV doses represent clones that are resistant to photosensitization, a phenomenon attributed to either inability to take up or metabolize P12, or resistance to the radiation-induced toxicity. Analysis of P12 uptake in the surviving clonal populations showed no significant difference as compared to the parental population. The results suggest that surviving cells reflect a phenotypic heterogeneity caused by variation in the physiological state such as the respective position in the cell cycle and are not genetically resistant mutants.

Cell Membrane↗
PubMed1987

Diagnosis of arylsulfatase A deficiency in intact cultured cells using a fluorescent derivative of cerebroside sulfate.

A fluorescent derivative of cerebroside sulfate (12-(1-pyrene)dodecanoyl-sphingosylgalactosyl-0-3-sulfate (P12-sulfatide) has been synthesized as a potential substrate for the determination of cerebroside sulfatidase (or arylsulfatase A) activity. It was administered into cultured human skin fibroblasts and thereby utilized for the diagnosis of arylsulfatase A deficiency. Cultured skin fibroblasts from normal individuals and healthy persons suffering from a pseudoarylsulfatase A deficiency (PD) degraded the P12-sulfatide, while in cells derived from a metachromatic leukodystrophy (MLD) patient it remained essentially intact. This contrasts with in vitro determinations of enzymatic activity, where the MLD or PD-derived arylsulfatase A exhibit similar deficiency, in spite of a profoundly different clinical course. Administration of the fluorescent sulfatide into the intact cells permitted a sensitive and rapid diagnosis of MLD and its distinction from the PD-phenomenon. This might be of particular importance for cases in which a rapid diagnosis is required and for prenatal diagnosis of fetuses from families afflicted with both MLD and pseudo-deficiency mutant genes.

Cell Line↗
PubMed1986

Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes.

Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.

Gaucher Disease↗
PubMed1986

Utilization of membranous lipid substrates by membranous enzymes: activation of the latent sphingomyelinase of hen erythrocyte membrane.

Previous studies demonstrated that hen erythrocytes have an inoperative, latent sphingomyelinase which is activated when the cells are hemolyzed in a hypotonic medium. Within minutes after hemolysis about 60-80% of the sphingomyelin (SPM) of the RBC "ghost" membrane was hydrolyzed. In this paper, expression of sphingomyelinase activity was further investigated. The percentage of total SPM hydrolyzed depended on the volume of the hypotonic hemolyzing buffer. Thus, suspending the erythrocytes in 4 vol of the buffer resulted in clumping of the hemolyzed "ghosts" and no hydrolysis of SPM. In comparison, suspension in 19 vol of the hypotonic buffer showed no clumping and sphingomyelinase activity was fully expressed. But centrifugation of the latter or, alternatively, addition of concanavalin A induced clumping and elimination of sphingomyelinase activity. Hen RBC could also be hemolyzed in an isotonic medium in the presence of Triton X-100, mellitin, halothane, and phospholipase C. Activation of the latent sphingomyelinase occurred at concentrations of these reagents which caused cell lysis. Hen RBC were dispersed in an isotonic medium containing glutaraldehyde (0.1%) or formaldehyde (10%). This rendered the cells resistant to hemolysis, even when subsequently dispersed in a hypotonic medium or water. But incubation of the "fixed" cells in a hypotonic or isotonic medium activated the enzyme, resulting in hydrolysis of 60% of the cellular SPM. In contrast, when glutaraldehyde was included in the hypotonic buffer, hemolysis occurred but sphingomyelinase activity was eliminated.

Animals↗