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Concanavalin A is visibly aggregated by low concentrations of sodium dodecyl sulfate, maximum aggregation being obtained at pH 4.6. Other denaturants, such as urea, guanidine hydrochloride, Triton X-100, cetyltrimethylammonium bromide, Tween 80, and Brij 35 are ineffective in promoting visible aggregation. The sodium dodecyl sulfate-induced aggregation of concanavalin A requires the presence of an intact, saccharide-ligand binding-site. Rapid and complete reversal of the detergent effect was achieved by use of saccharides which bind to the lectin. Such compounds as tryptophan and o-nitrophenyl beta-D-galactopyranoside did not inhibit the aggregation of concanavalin A by sodium dodecyl sulfate, suggesting that the detergent does not bind the hydrophobic pocket on the surface of the protein. The results suggest that concanavalin A may have an additional, ligand-binding site which is metal-dependent and which can be modified by the addition of a saccharide ligand.

Sera from 9 persons with either biopsy-proven alcoholic liver disease or a history of chronic, excessive ethanol consumption were analyzed for their content of various hydrolases. Compared to controls, significant elevations in the following enzyme activities were seen in sera from the patient population: acid phosphatase (2.0-fold), beta-glucuronidase (2.1-fold), hexosaminidase (1.4-fold), and alpha-L-fucosidase (2.3-fold). In addition, alpha-mannosidase activity, previously reported to be unchanged in cases of hepatic cirrhosis [Reglero et al., Clinica chim. Acta 130: 155-158], (1980) was found to be significantly increased (p less than 0.001) when assays were performed at acid (pH 4.5) or intermediate (pH 5.5) hydrogen ion concentrations. Fractionation of sera on DEAE-Sephadex columns showed that the increase in alpha-mannosidase activity in the serum of patients with alcoholic liver disease was due to increases in the level of at least one 'acid alpha-mannosidase' and two intermediate pH optimum alpha-mannosidases. The general increase in the activity of a group of glycosidases is consistent with a hypothesis involving decreased clearance of glycoproteins from the blood of persons with hepatic cirrhosis.

Identification of biochemical changes induced by ethanol ingestion would aid in the diagnosis and management of many alcohol-related problems in man. In this paper we identify a pH 5.5 alpha-mannosidase activity in the rat which is affected by chronic ethanol consumption. Chronic (16 wk) ingestion of alcohol (36% of calories) causes the activity of this alpha-mannosidase (thought to be the cytosolic alpha-mannosidase) in liver to decrease by 50%. We hypothesize that this deficiency of (pH 5.5) alpha-mannosidase activity may account for the reduced rate of secretion of glycoproteins by livers of alcohol-fed rats reported by other investigators (Volentine et al, Hepatology 1987;7:490-495).

Sequential extraction of human spleen membranes with sodium cholate and n-butanol removes endogenous lipids and renders glucocerebrosidase activity dependent upon exogenous acidic lipids (e.g., phosphatidylserine, gangliosides) and a heat-stable activator protein (HSF). In the present report, we show that two previously untested lysosomal acidic lipids, namely sulfogalactocerebroside and bis-(monoacylglyceryl)-phosphate (BMP), also activate normal human glucocerebrosidase. In addition, sulfogalactocerebroside also markedly enhanced the activity of glucocerebrosidase isolated from a patient with type 1 (non-neuronopathic) Gaucher's disease, resulting in a specific activity which was 60-80% that of control glucocerebrosidase. Furthermore, when the sulfolipid was used as the activator, glucocerebrosidase from the type 1 patient was 30 times more active than the corresponding glucocerebrosidase from a person with type 2 (neuronopathic) Gaucher's disease. In contrast, the two BMPs, one rich in C26 saturated fatty acid and another rich in C18 unsaturated fatty acids, were relatively poor activators of both mutant glucocerebrosidases while providing excellent reconstitution of control activity.

1. More than 90% of the total acid phosphatase activity in a sonicate of L. donovani promastigotes is contained in a particulate fraction (200,000 X g 30 min). The enzyme can be quantitatively extracted and solubilized with the aid of Triton X-100 (0.2 g/100 ml) and purified over 200-fold with 54% yield by chromatography on DEAE-Sephadex, QAE-Sephadex, Sepharose 4B and concanavalin-A Sepharose. 2. The phosphatase is a true acid hydrolase (pH optimum, 5.0-5.5) and has a rather broad substrate specificity; it will catalyze the hydrolysis of 4-methylumbelliferylphosphate, thymolphthalein diphosphate, pyridoxal phosphate, fructose 1,6-diphosphate, glucose 6-phosphate, glucose 1-phosphate, ADP and AMP. 3. It is a large (170,000 daltons in the presence of Triton X-100), stable and acidic enzyme (pI = 4.1) that has the electrophoretic mobility of a type zero or type 1 isoenzyme in acid (pH 4.3) polyacrylamide gels. 4. The enzyme is inhibited by sodium fluoride, 2-mercaptoethanol and mumolar amounts of a number of polyanionic molybdenum and heavy metal complexes that include the following: [C(NH2)3]4[(C3H7O3PO3)2Mo5O15] X 3H2O, [C(NH2)3]2[(C6H5)2AsMo4O15H] X H2O, (NH4)4[SiMo12O40] X H2O and (NH4)6[P2Mo18O62] X 9H2O. 5. L. donovani promastigotes contain very low levels of 10 other acid pH optimum hydrolytic enzymes, with the exception of modest levels of alpha-fucosidase.

Biochemical studies of middle ear effusions have demonstrated generally higher levels of certain hydrolytic and oxidative enzymes in mucoid fluids when compared to serous. We have extended these studies by analyzing middle ear effusions for the content of a large number of lysosomal hydrolases. The mean specific activity for alpha-glucosidase in mucoid fluids was found to be ten times that for serous fluids while alpha-mannosidase, beta-glucuronidase, hexosaminidase, acid phosphatase, beta-galactosidase, alkaline phosphatase, and lactate dehydrogenase were found to be three to five times greater in mucoid than serous effusions. In this study the specific enzyme activities for lysosomal hydrolases from purulent effusions were found to be intermediate between the activities in serous and mucoid effusions. No significant correlation was found between the specific activities of lysosomal hydrolases and the presence or absence of bacteria in mucoid or serous middle ear effusions. The hexosaminidase isozyme distribution was found to be identical for serous and mucoid fluids and similar to that found in human serum. However, the isozyme pattern of beta-glucuronidase in mucoid effusions was significantly different than that in normal human serum as mucoid fluids contain a large amount of an anionic isoenzyme of beta-glucuronidase that is barely detectable in human serum.

The in situ lipid activator of rat liver glucocerebrosidase was investigated. Rat liver lysosomes were purified (42.9-fold relative to the crude homogenate) by sequential isopycnic centrifugation in sucrose and metrizamide gradients. Lipids were extracted with chloroform:methanol (2:1) and phospholipids were separated by one-dimensional thin-layer chromatography. The phospholipid content of the lysosome preparation was 0.28 mumol lipid phosphorus/mg protein. Phosphatidylcholine was present as the major nonacidic phospholipid (39.3%). Of the acidic phospholipids, phosphatidylinositol and phosphatidylserine were present in the greatest amounts (12.0 and 19.7%, respectively). The resolved phospholipids were tested separately and in the presence of a heat-stable factor from Gaucher spleen for their ability to reconstitute butanol-delipidated rat liver glucocerebrosidase activity. Alone or in the presence of the heat-stable factor, phosphatidylserine and phosphatidylinositol were the most effective activators of glucocerebrosidase. Bis(monoacylglyceryl) phosphate derived from rat liver tritosomes or rabbit lung macrophages was also effective in reconstituting beta-glucosidase activity.

A procedure is described for the preparation of extensively purified beta-D-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the beta-glucosidase in the high speed supernatant (100 000 X g, 90 min) fraction of rat kidney homogenate is 700-fold greater than that in the same fraction from heart, skeletal muscle, lung, spleen, brain or liver. beta-Glucosidase activity co-chromatographs with beta-D-galactosidase, beta-D-fucosidase, alpha-L-arabinosidase and beta-D-xylosidase activities through the last four column steps of the purification and their specific activities are 0.26, 0.39, 0.028 and 0.017 relative to that of beta-glucosidase, respectively. The specific activity of the apparently homogeneous beta-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-beta-D-glucopyranoside per mg protein per h. All five glycosidase activities possess similar pH dependency (pH optimum, 6--7) and heat lability, and co-migrate on polyacrylamide disc gels at pH 8.9 (RF, 0.67). beta-Glucosidase acitivity is inhibited competitively by glucono-(1 leads to 5)-lactone (KI, 0.61 mM) and non-competitively by a variety of sulfhydryl reagents including N-ethylmaleimide, p-chloromercuribenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid), and iodoacetic acid. Although the enzyme will release glucose from p-nitrophenyl and 4-methylumbelliferyl derivatives of beta-D-glucose, it will not hydrolyze xylosyl-O-serine, beta-D-glucocerebroside, lactose, galactosylovalbumin or trehalose. The enzyme consists of a single polypeptide chain with a molecular weight of 50 000--58 000, has a sedimentation coefficient of 4.41 S and contains a relatively large number of acidic amino acids. A study of the distribution of beta-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convoluted tubule. The enzyme is also present in relatively large amounts in the villus cells, but not crypt cells, of the intestine. The physiological substrate and function of the enzyme are unknown.

Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include beta-glucosidase, beta-galactosidase, beta-fucosidase, alpha-mannosidase, hexosaminidase, arylsulfatase A, and beta-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly "acid" hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: pH optima, 5.5; Km (4-methylumbelliferyl phosphate), 0.60 mM; molecular weight (estimated by gel filtration chromatography), 92,000; inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?

From 330 ml of rat serum, 222 mg of alpha-1-antitrypsin have been isolated and purified with an overall yield of approximately 20%. The preparation was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sedimentation equilibrium centrifugation, and immunoelectrophoresis. Rat alpha-1-antitrypsin exhibited Mr = 47,000 +/- 1,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 1,000 by equilibrium ultracentrifugation; the sedimentation coefficient (s20,w) was 3.29. Rat alpha-1-antitrypsin exhibited a trypsin-combining ratio (moles of trypsin inhibited/mol of alpha-1-antitrypsin) of 0.88. Rat alpha-1-antitrypsin showed significant differences in amino acid composition when compared to human alpha-1-antitrypsin, particularly in lysine, glutamic acid, arginine, methionine, and tyrosine content. Rat alpha-1-antitrypsin contains 14.3 residues/mol of N-acetylglucosamine, 5.0 residues/mol of mannose, 4.2 residues/mol of galactose, and 5.8 residues/mol of sialic acid. Monospecific antibody produced in a rabbit against our purest preparation of rat alpha-1-antitrypsin does not cross-react immunologically against human, calf, fetal calf, mouse, or chicken sera. The availability of a pure preparation of rat alpha-1-antitrypsin as well as the specific alpha-1-antitrypsin antibody will facilitate studies on the biosynthesis and secretion of this important protease inhibitor in an appropriate animal model.

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.

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.

We have isolated from guinea-pig liver a broad-specificity beta-glucosidase of unknown function that utilizes as its substrate non-physiological aryl glycosides (e.g. 4-methylumbelliferyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside). The present paper documents that this enzyme can be inhibited by various naturally occurring glycosides, including L-picein, dhurrin and glucocheirolin. In addition, L-picein, which acts as a competitive inhibitor of the broad-specificity beta-glucosidase (Ki 0.65 mM), is also a substrate for this enzyme (Km 0.63 mM; Vmax. 277,000 units/mg). Heat-denaturation, kinetic competition studies, chromatographic properties and pH optima all argue strongly that the broad-specificity beta-glucosidase is responsible for the hydrolysis of both the non-physiological aryl glycosides and L-picein. This paper demonstrates that beta-glucosidase can catalyse the hydrolysis of a natural glycoside, and may provide a key to understanding the function of this enigmatic enzyme. A possible role in the metabolism of xenobiotic compounds is discussed.

The activities of a number of lysosomal hydrolases were determined in sera from 100 patients with cystic fibrosis (CF), age 2-35 yr, and age-matched controls: beta-hexosaminidase activity was significantly elevated (p less than 0.005) in CF patients from all age groups. alpha-Mannosidase activity was increased only in the older CF patients (greater than 13 yr). The following enzyme activities were not altered in CF serum: alpha-fucosidase, beta-glucuronidase and acid phosphatase. The abnormal patterns of serum alpha-mannosidase and beta-hexosaminidase in CF cannot be explained by pancreatic disease or undernutrition, since serum values of these hydrolases in patients with anorexia nervosa or acute pancreatitis were not altered. However, the altered activities of the alpha-mannosidase and beta-hexosaminidase were proportional to the degree of pulmonary insufficiency in the CF group, indicating that these changes are probably a secondary consequence of the primary disease process. Except for beta-hexosaminidase, because differences in the serum hydrolases in CF do not become apparent until the second decade of life, determinations of lysosomal enzyme activities in serum will probably be of little diagnostic value.

The physician who diagnoses Gaucher's disease should take advantage of the noninvasive method that analyzes WBCs for residual beta-glucocerebrosidase and beta-glucosidase activities. When this method is carried out in conjunction with the measurement of serum acid phosphatase levels, a bone marrow examination may be unnecessary. With this method, we studied an adult who had mild splenomegaly and abdominal pain. When bone marrow was finally obtained subsequent to diagnosis by the enzymatic analysis, the deposits that are specifically formed in Gaucher's disease were easily demonstrated by electron microscopy. We believe that these methods are more specific for the diagnosis of Gaucher's disease than is the light microscopic finding of bone marrow cells that have abundant and striated cytoplasm.

Three fluorometric leukocyte beta -glucosidase assays were compared for their ability to diagnose Gaucher's disease and identify carriers of the disorder: the acid beta-glucosidase assay of Beutler and Kuhl [2], a pH 5.5-sodium taurocholate-dependent assay and a new procedure which employs conduritol B epoxide, an active-site specific inhibitor of glucocerebrosidase. All three assays unambiguously identified patients with Gaucher's disease. With regard to identifying carriers the bile salt dependent assay of Peters et al. and the conduritol B epoxide-dependent procedure gave the greatest discrimination between the mean beta-glucosidase values for the control and heterozygote samples when evaluated using Student's t test. The most reliable assay for the identification of the carrier state was the conduritol B epoxide-dependent procedure which can be expected to provide the fewest false negative results when classifying heterozygotes (5%). However, the fact that none of these methods will completely separate control and heterozygote samples indicates that their use in screening programs will result in a significant number of incorrect assignments.

Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms of its ability to hydrolyse the water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity in a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 microM; CMC3, 60.7 microM. We designate the aggregates formed at these CMCs as primary, secondary and tertiary micelles respectively. From the results of kinetic studies performed at various sulphatide concentrations (0.012-248 microM), we found that sulphatide monomers (less than 3 microM) decreased the Km (for MUGlc) of control glucocerebrosidase from 11 to 4.6 mM, and lowered the Vmax. 2-fold. However, secondary and tertiary micelles were required for expression of high control glucocerebrosidase activities. Glucocerebrosidase prepared from the spleen of a patient with non-neuronopathic type 1 Gaucher's disease exhibited a very low Km (2.8 mM) even in the absence of exogenous lipid, and sulphatide monomers had no effect on the mutant enzyme's Km or Vmax. However, secondary or tertiary micelles markedly increased the Vmax. of the type 1 glucocerebrosidase to 60% of the corresponding control enzyme value. In contrast, for the glucocerebrosidase of the neuronopathic type 2 case, although sulphatide decreased the Km from 9.2 to 1.7 mM, the Vmax. never reached more than 5% that of the control enzyme, even at high concentrations of sulphatide. In addition, we found that secondary and tertiary sulphatide micelles enhanced the rate of inactivation of all three glucocerebrosidase preparations by chymotrypsin. Collectively, these results indicate the presence of two sulphatide-binding sites on glucocerebrosidase: one that enhances substrate binding, and another that enhances catalysis.

Although the primary genetic defect in all individuals with Gaucher's disease is a deficiency in glucocerebrosidase activity, the finding of marked elevations in splenic and serum acid phosphatase activity is almost as consistent a finding. Gaucher spleen and serum contain at least two forms of acid phosphatase that can be readily separated by chromatography on columns containing the cation exchange resin Sulphopropyl Sephadex. The major species of acid phosphatase (designated SP-I) contained in Triton X-100 (1% v/v) extracts of Gaucher spleen accounts for 65%--95% of the total activity and has the following properties: (1) it does not bind to the cation exchange column; (2) it exhibitis a pH optimum of 4.5--5.0; (3) it is inhibited by sodium fluoride (15 mM), L(+)-tartaric acid (20 mM), and beta-mercaptoethanol (2.1 M), and (4) it is resistant to inhibition by sodium dithionite (10 mM). The minor acid phosphatase activity (designated SP-II) present in extracts of Gaucher spleen has properties similar to those of the major species of acid phosphatase activity contained in serum from patients with Gaucher's disease: (1) it binds firmly to cation exchange columns (eluted by 0.5 M sodium chloride); (2) it exhibits a pH optimum of 5.0--6.0; (3) it is inhibited by sodium fluoride and sodium dithionite; and (4) it is resistant to inhibition by beta-mercaptoethanol (2.1 M) and L(+)-tartaric acid (20 mM). In addition, a second form of acid phosphatase that is tartrate resistant was found to be elevated in Gaucher serum. This form of serum acid phosphatase did not bind to Sulphopropyl Sephadex, was found to be significantly resistant to beta-mercaptoethanol (2.1 M), and was only partially inhibited by sodium dithionite (10 mM). The findings reported here indicate that at least three distinct forms of acid phosphatase activity are elevated in Gaucher's disease. Furthermore, the minor acid phosphatase activity contained in spleen homogenates has properties very similar to those of the major acid phosphatase activity observed to be present in serum of patients with Gaucher's disease. These data indicate that simple spleen spillage cannot account for the increased levels of serum acid phosphatase in patients with Gaucher's disease.

To date, enzymatic diagnosis of Gaucher's disease via a fluorometric assay procedure which utilizes 4-methylumbelliferyl-beta-D-glucopyranoside as a substrate has not been possible when liver serves as the source of enzyme since currently employed fluorometric procedures cannot adequately differentiate between a broad-specificity beta-glucosidase and lysosomal glucocerebrosidase activities in crude extracts of liver. Incorporation of conduritol-beta-epoxide into the incubation medium for the fluorometric assay allows one to selectively measure the glucocerebrosidase activity present in a given liver extract. In five cases of Gaucher's disease this revised fluorometric procedure proved as effective as the assy procedure which utilizes authentic, radiolabeled glucocerebroside as the substrate in demonstrating a deficiency of glucocerebrosidase activity in liver.

1. A third beta-glucosidase from human liver has been isolated using a mild (0.02-0.10%) Triton X-100 extraction of the exhaustively washed high speed (200,000 X g, 30 min) particulate fraction, QAE-Sephadex and concanavalin A-Sepharose chromatography. This new beta-glucosidase, referred to as TX beta-glucosidase, possesses a distinctive set of chemical properties such that it is similar to both, glucocerebrosidase and cytoplasmic beta-glucosidase, but it is not identical to either enzyme. 2. The TX beta-glucosidase hydrolyzes glucocerebroside as well as the beta-D-glucose, beta-D-galactose, beta-D-fucose, beta-D-xylose and alpha-L-arabinose derivatives of 4-methylumbelliferone. Like the cytoplasmic beta-glucosidase, the TX beta-glucosidase is inhibited by bile salts, and unaffected by conduritol B epoxide and heat stable activator protein. 3. All three beta-glucosidases were inhibited by N-hexylpsychosine, and all showed the same, mixed type inhibition kinetics, indicating a common hydrophobic binding site in all three enzymes. 4. The TX beta-glucosidase, which constitutes only a few percent of the total beta-glucosidase activity of human liver, is absent from liver from two cases of neurologic Gaucher disease and present in reduced amounts in a third case with CNS disease. Liver from a case of type 1 Gaucher disease contained normal amounts of the TX beta-glucosidase.

We have shown that the protein-deficient weanling rat fed a 3% casein diet, within 2 to 4 wk, exhibits marked changes in serum lysosomal hydrolases similar to those observed in children suffering from protein-calorie malnutrition: serum hexosaminidase, alpha-mannosidase, and beta-glucuronidase activities increase 3-fold, 2-fold, and 50%, respectively, whereas the acid phosphatase levels decrease by 50%. Rehabilitation of the protein-deficient animals with a diet containing 25% protein (i.e., casein) results in a rapid restoration of the plasma lysosomal hydrolase profiles to normal in less than 1 wk. The specific activities of various tissue lysosomal enzymes change significantly in the protein-deficient animals; however, no overall consistent pattern of change is apparent. In general, the greatest number of changes in lysosomal enzymes occurs in the kidney, whereas the brain exhibits the smallest differences between experimental and control animals in this regard. Perfusion experiments have shown that the rate of release of lysosomal enzymes from livers of rats fed the protein-deficient diet is profoundly altered when compared to that of control animals. Studies of the variation of enzyme secretion with time have demonstrated that the rate of secretion of hexosaminidase by the liver remains low and then rises markedly (3-fold) after the animals have been consuming the 3% casein diet for 16 days. In contrast, the secretion of both acid phosphatase and beta-glucuronidase is markedly depressed in the early phase of protein malnutrition (i.e., 7 to 16 days), and then increases greatly by the 3rd wk. These results demonstrate that changes occur in the rate of secretion of lysosomal enzymes by the liver during the course of experimental protein malnutrition.

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, histone-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [gamma-32P]ATP and Mg2+ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [gamma-32P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA+). The PNA- and PNA+ forms of L. major both phosphorylated a 50-kDa protein when incubated with [gamma-32P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

In an effort to facilitate studies of the reaction involved in the removal of fatty acids from acyl proteins, we have synthesized an octanoic acid ester of doubly blocked serine, specifically octanoyl N-carbobenzoxy-L-serine-benzyl ester (octanoyl boc-serine), and used it as a substrate to guide the purification of an esterase from rat lung. The esterase was purified 228-fold by column chromatography on DE-52 cellulose, hydroxylapatite, octyl-Sepharose, and concanavalin A-Sepharose and by HPLC gel filtration. The final enzyme preparation ran as a single 77,000-Da band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited a single symmetrical peak (sedimentation coefficient, 4.5 S) when centrifuged through a sucrose density gradient (empirical Mr, 63,000). The esterase is an acidic protein, pI 4.1, and is very active against p-nitrophenyl esters comprised of C4-C14 fatty acids; the highest specific activity (26.5 mumol/min/mg) was obtained using p-nitrophenyl caprylate as substrate. The pH optimum of the lung esterase is near 8.0 and the activity on octanoyl boc-serine is maximum when 0.3% (w/v) Myrj-52 is included in the assay medium. The activity of the esterase is not dependent on calcium ions. The enzyme does not remove acyl groups from the G-protein of vesicular stomatitis virus or the proteolipid of bovine brain. The possible role of the esterase in the metabolism of acylated proteins is considered.

A cytoplasmic beta-glucosidase has been isolated and purified 9,000-fold to homogeneity from the liver of a case of type 1 Gaucher's disease to a specific activity of 400,000 nmol/h/mg of protein. Although markedly elevated above control levels in this case of adult Gaucher's disease, the activity of this cytosolic liver enzyme was found to be markedly deficient in two cases of neurologic Gaucher's disease. The purification scheme employs QAE-Sephadex, DE52 cellulose, CM-Sephadex, hydroxylapatite, and Cibacron blue-Sepharose chromatography, and preparative isoelectric focusing. The beta-glucosidase preparations isolated from the liver of the case of adult Gaucher's disease and control liver have similar physical properties. Both enzymes have a molecular weight of approximately 53,000, sw,20 of 4.3, pI of 4.5-4.6, a pH optimum between 5 and 6, and a high affinity for 4-methylumbelliferyl-beta-D-glucopyranoside (Km = 0.06-0.07 mM). The enzymes from both sources also have a broad specificity and will hydrolyze the 4-methylumbelliferyl derivatives of beta-D-galactose, beta-D-fucose, beta-D-xylose, and alpha-L-arabinose in addition to several aryl-galactosides and steroid-glucosides. The cytoplasmic beta-glucosidase will not hydrolyze glucocerebroside and shows no cross-reactivity with antibodies prepared against lysosomal glucocerebrosidase. Both cytoplasmic beta-glucosidase and glucocerebrosidase will hydrolyze 17 beta-estradiol-17'-beta-D-glucose, and the activity of both enzymes on this substrate is increased more than 15-fold in the presence of the Gaucher spleen heat-stable factor. The role of this cytoplasmic beta-glucosidase in the etiology of Gaucher's disease and its possible relationship to lysosomal glucocerebrosidase are discussed.

We report a new assay for the detection of individuals heterozygous and homozygous for Gaucher's disease which requires relatively small samples of whole blood (0.3 ml), and which determines 4-methylumbelliferyl-beta-D-glucopyranoside:beta-glucosidase activity under conditions optimal for the determination of leukocyte glucocerebroside:beta-glucocereborsidase activity. The procedure involves the preparation of a leukocyte pellet from 50 mul of whole blood by hypotonic lysis of erythrocytes, followed by assay of beta-glucosidase activity at pH 5.5 in the presence of sodium taurocholate (0.6 g/100 ml). The methods described may also prove to be useful for the diagnosis of other diseases of enzyme deficiency which use fluorogenic substrates and leukocytes as a source of enzyme, such as Fabry's disease, Tay-Sachs disease, and generalized gangliosidosis.

The description in 1965 of glucocerebroside: beta-glucosidase as the enzymic defect in Gaucher's disease stimulated considerable research interest and effort toward establishing rapid, reliable, and inexpensive enzymic assays for diagnostic purposes and carrier detection. Here, we consider some of the methods currently in use in which the substrate is the synthetic glucoside, 4-methylumbelliferyl-beta-D-glucopyranoside, and leukocytes and fibroblasts are the sources of enzyme. We also consider the concepts of the "acid beta-glucosidase" and multiple forms of beta-glucosidase that have been proposed to explain the effectiveness of the fluorometric assays. Finally, we analyze the limitations of each method and discuss the difficulties involved in instituting heterozygote screening programs in the general population.

In a 1969 prisoner volunteer study of malaria transmission by blood inoculation, six persons were subinoculated sequentially, and acute hepatitis occurred in the last four (sequential study). Subsequently, another 15 volunteers receiving malaria-rich blood from 14 different donors were followed prospectively (prospective study), and hepatitis developed in six. Incubation periods were shorter but serum transaminase levels were higher for the cases of hepatitis occurring in the sequential study than in the prospective study. Although the illnesses were clinically mild, elevations in transaminase levels persisted for more than six months in five and fluctuating transaminase activities were observed in nine of the 10 affected persons. In addition, an 11th prisoner developed sporadic hepatitis. Neither known human hepatitis viruses nor malaria could be implicated in these cases, which were classified as non-A, non-B (NANB) hepatitis. The data suggested that the viremia of short-incubation NANB hepatitis may begin within the first week after inoculation, confirmed that NANB hepatitis may be transmitted either percutaneously or nonpercutaneously, and provided further evidence that there is more than one NANB agent.

The activity of the lysosomal enzymes acid phosphatase, beta-glucuronidase, alpha-mannosidase and hexosaminidase were determined in CSF obtained from patients with proven bacterial meningitis and from patients with various other diagnoses. The mean value for CSF beta-glucuronidase from bacterial meningitis was elevated 73-fold when compared to the aggregate mean of all control groups. Acid phosphatase and alpha-mannosidase means were 26-fold and 33-fold elevated respectively while hexosaminidase was threefold elevated. Measurement of CSF acid phosphatase and beta-glucuronidase should prove a rapid useful test in establishing the diagnosis of bacterial meningitis. Chromatography of CSF samples on DEAE Sephadex allowed the resolution of hexosaminidase and beta-glucuronidase into individual isozymes. The ratio of hexosaminidase A to hexosaminidase B was generally higher in CSF from patients with bacterial meningitis but was very variable. The isozyme distribution for beta-glucuronidase was identical to that found in serum and no differences in pattern were found between patients and control subjects.

The molecular basis for the accumulation of a substance which displays the immunological reactivity of alpha-1-antitrypsin within vesicles of liver parenchymal cells of individuals with hepatic cirrhosis and serum alpha-1-antitrypsin deficiency remains unclear. We recently reported that serum from a patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis was substantially deficient in sialyltransferease (EC 2.4.99.1) an enzyme which transfers sialic acid from cytidine 5'-monophosphate-N-acetylneuraminic acid to a variety of asialoglycoprotein acceptors. In the present report we have extended these studies to include serum from five additional patients with alpha-1-antitrypsin deficiency and juvenile hepatic cirrhosis as well as a liver specimen obtained at autopsy of one of these patients. We find the sialytransferase activity in serum from six patients with alpha-1-antitrypsin deficiency and hepatic cirrhosis to be 50% of healthy pediatric control values and 30% of pediatric patients with liver disease. However, serum from family members homozygous for alpha-1-antitrypsin deficiency but without hepatic cirrhosis, and serum from patients with a variety of other kinds of liver disease, failed to exhibit the marked sialytransferase deficiency. Similar assays carried out on a homogenate of a liver sample from one patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis indicated that the deficiency of sialyltransferase activity was not demonstrable in liver. Furthermore, a comparative kinetic analysis of serum and liver sialytransferase in normal and afflicted individuals failed to detect differences in substrate affinities which might account for a decrease in functional sialyltransferase capacity in individuals with alpha-1-antitrypsin deficiency and hepatic cirrhosis. These observations suggest that the serum sialyltransferase deficiency in such patients probably arises after chronic and extensive liver disease involving hepatic accumulation of alpha-1-antitrypsin rather than the enzyme deficiency being the primary cause of the hepatic cirrhosis and alpha-1-antitrypsin deficiency.

Protein kinases I (PK I) and II (PK II) were purified 253- and 13.5-fold, respectively, from an extract of sonically disrupted cells of Legionella micdadei by ion-exchange chromatography on QAE-Sephadex, by histone affinity chromatography, and by HPLC-gel filtration chromatography. Both enzymes catalysed the phosphorylation of calf thymus histones, with a Km of 2.7 mg ml-1 for PK I and 2.9 mg ml-1 for PK II. Histone H2b was the best protein kinase substrate for both PK I and PK II. The pH optima were 6.8 and 7.0 for PK I and PK II respectively. The Km for ATP was 0.29 mM for PK I and 0.33 mM for PK II. PK II activity was stimulated by either cAMP or cGMP, whereas PK I was inhibited by both cyclic nucleotides. The activity of PK I was unaffected by addition of calmodulin, diacylglycerol and mixtures of Ca2+ and acidic phospholipids, but these additions increased PK II activity threefold. The activity of PK II was stimulated by spermine and spermidine, but PK I was inhibited by these compounds. PK I and PK II were both strongly inhibited by heparin.

Analyses of Streptococcus pneumoniae culture filtrates and middle ear effusions (MEE) containing S pneumoniae for various hydrolytic enzymes have demonstrated substantial levels of neuraminidase activity when measured employing a sensitive fluorometric assay. S pneumoniae neuraminidase exhibits optimum activity near neutral pH (6.0 to 6.5), and catalyzes the cleavage of sialic acid residues from glycoproteins, gangliosides and mucopolysaccharides. S pneumoniae begins secreting large amounts of neutral neuraminidase (mean [means] = 43.3 units/mL culture filtrate) when cells enter the stationary phase. Nearly all (96%) human chronic MEEs yielding positive cultures for S pneumoniae contain neuraminidase activity (means = 0.200 units/mg protein), while only 21.1% to 45.5% of all other effusions contain the enzyme. Middle ear effusions obtained from S pneumoniae infected-chinchillas contained large amounts of neuraminidase activity (approximately 200 units/mL), which decayed exponentially in vivo with an apparent half-life of 8 1/2 days. Three neuraminidase isoenzymes (designated I-III) were identified in S pneumoniae culture filtrates, as well as in MEEs from chinchillas infected with the organism, using a combination of ion-exchange and gel filtration chromatography. With 4-methylumbelliferyl-N-acetylneuraminic acid serving as substrate, preparation I from both culture filtrates and MEEs was characterized by a high Michaelis constant (Km), while forms II and III had low Km values. Preferred substrates were orosomucoid and neuramin-lactose; gangliosides, thyroglobulin, and bovine submaxillary mucin were poorer substrates.