Mic N. Hamers

Phagocytosing human neutrophils inactivate their own granular enzymes.

During phagocytosis, neutrophils generate reactive oxygen metabolites and release lysosomal enzymes into the extracellular medium. We have investigated the possibility that these enzyme are inactivated by the oxygen compounds. Phagocytosing neutrophils from 12 patients with chronic granulomatous disease, which do not generate these oxygen metabolites, released two to three times more activity of lysozyme and beta-glucuronidase than did normal neutrophils. This difference proved to be due to a decrease of approximately 20% of the total activity of these enzymes in normal neutrophils, but not in neutrophils of patients with chronic granulomatous disease. This inactivation of enzymes took place during phagocytosis of opsonized zymosan particles as well as during stimulation of normal cells with phorbol myristate acetate. The inactivation was not due to formation of inhibitors. The lysosomal enzymes were not activated when the neutrophils were stimulated under anaerobic conditions. Addition of catalase, superoxide dismutase, or albumin gave no protection against the oxidative damage; reduced glutathione gave partial protection. The oxidative inactivation was more pronounced in the presence of azide. Measurement of the activity and the amount of protein of acid alpha-glucosidase in the cells showed that the specific activity of this enzyme decreased by approximately 50% during 30 min of phagocytosis. This indicates that the inactivation of the lysosomal enzymes takes place in the phagolysosomes, before the enzymes have leaked into the extracellular medium.

Human eosinophil peroxidase: a novel isolation procedure, spectral properties and chlorinating activity.

Like neutrophils, eosinophils contain a high concentration of a peroxidase that may have enzymic activity which is associated with the antiparasitic function of these cells [ 11. In neutrophils it has been demonstrated that the peroxidase (myeloperoxidase) is involved in the killing of micro-organisms (reviewed in [2]). This antimicrobial activity of myeloperoxidase is probably due to the ability of this enzyme to oxidize Clwith HzOz to the reactive hypochlorous acid (HOCl) [3-51. It is not known whether human eosinophil peroxidase can catalyse the same reaction. It was recently shown [6], however, that eosinophil peroxidase partially purified from guinea pig eosinophils is bactericidal when combined with Hz02 and Cl-. Studies on human eosinophil peroxidase are hampered since eosinophils can only be obtained in significant amounts from blood from patients with eosinophilia. However, in order to be able to purify the peroxidase from these eosinophils and to study the enzyme in some more detail, sufficient patient blood must be available, which is generally not the case. In addition, it is conceivable that the properties of patient eosinophils, and the enzymes in these cells, differ from those normally foundin blood circulation. As shown in [7] the amount of haem iron derived from the peroxidase in an eosinophil is -4-times higher than the concentration of myeloperoxidase in a neutrophil. Since white blood cells of normal donors contain 3-S% eosinophils, 12-20% of the total peroxidase (on the basis of haem iron) in a homogenate of white cells must be due to eosinophil peroxidase. From 100 1 human blood 200 mg pure myeloperoxidase can be obtained [8]; it should therefore be pos-

The identity of α-galactosidase B from human liver

Abstract 1. 1. The identity of α-galactosidase B (α- d -galactoside galactohydrolase, EC 3.2.1.22), the minor α-galactosidase isoenzyme present in normal human tissues and the component responsible for the residual α-galactosidase activity in patients with Fabrys disease, was investigated. For this investigation, α-galactosidase B was purified from normal human liver. 2. 2. Purified α-galactosidase B contains α-glucosidase, α-xylosidase, N-acetyl-αgalactosaminidase and α-galactosidase activity, as measured with p-nitrophenyl glycosides as substrates. 3. 3. Incubation of purified α-galactosidase B with an antiserum against the same preparation resulted in a reduction of both α-galactosidase and N-acetyl-αgalactosaminidase activities. The extent of the reduction was dependent on the amount of antiserum added, an identical titration curve being obtained with both activities. On the other hand, the α-glucosidase activity was not affected by incubation with the antiserum and α-xylosidase was inactivated by incubation either with antiserum or with normal serum. 4. 4. Incubation of purified α-galactosidase B at 50°C for 4 h led to about 34% inactivation of both α-galactosidase and N-acetyl-α-galactosaminidase. 5. 5. The Km of purified α-galactosidase B for p-nitrophenyl-α-galactoside (about 20 mM) is higher than that for p-nitrophenyl-N-acetyl-α-galactosaminide (about 1 mM). The maximal velocity with the latter substrate is about 2.3-fold higher than that with the former. 6. 6. When purified α-galactosidase B is incubated with p-nitrophenyl-α-galactoside and p-nitrophenyl-N-acetyl-α-galactosaminide together, the rate of p-nitrophenol formation is considerably less than the sum of the rates observed when the substrates are added singly. 7. 7. It is concluded that the α-galactosidase and N-acetyl-α-galactosaminidase activities in purified α-galactosidase B are due to the same protein containing a single catalytic site.

Characterization of α-galactosidase isoenzymes in normal and fabry human-Chinese hamster somatic cell hybrids

SummaryThe α-galactosidases in normal man-Chinese hamster somatic cell hybrids were investigated with antibodies specific for human α-galactosidase A and antibodies specific for Chinese hamster α-galactosidase. It was found that an isoenzyme in hybrid cells, which has an electrophoretic mobility between that of human α-galactosidase A and Chinese hamster α-galactosidase, contains immunologic determinants of both human and Chinese hamster origin, suggesting that it is a heteropolymeric molecule. Moreover, the locus for human α-galactosidase, which was found to be X-linked, is the locus coding for α-galactosidase A. Hybrids isolated after fusion of Chinese hamster cells with cells of a patient with Fabrys disease did not express human α-galactosidase A or the heteropolymeric molecule even in the presence of the active human X chromosome, indicating that the deficiency of α-galactosidase A in Fabrys disease is probably due to a mutation in a structural gene resulting in the inability to form immunologically detectable and functionally active molecules of α-galactosidase A.

Characterization of antibodies against ceramidetrihexoside and globoside

Abstract 1. When a carrier-hapten complex was used to raise antibodies in rabbits against ceramidetrihexoside, an antiserum was obtained in which the activity against the carrier could not be removed by absorption with the carrier without loss of the anti-hapten activity. 2. Antibodies against ceramidetrihexoside were raised in rabbits by immunizing them with a mixture of bovine serum albumin and the glycolipid according to Hakomori (1972). It was found that these antibodies cross-reacted with digalactosylceramide. but not with lactosylceramide or digalactosyldiglyceride. No cross-reactivity was observed with globoside in an artificial membrane system but a slight cross-reactivity was found with globoside absorbed on cholesterol microcrystals. A possible explanation for the latter phenomenon is discussed. From inhibition studies with different sugar compounds combined with the cross-reactivity studies, it can be concluded that anti-ceramidetrihexoside antibodies have a high and strict specificity for a terminal galactosyl α(1→ 4)galactosyl group. 3. Antibodies raised in rabbits against globoside by the method of Hakomori (1972) did not cross-react with the other glycolipids tested. The characteristics of these antibodies are compared with those of the anti-ceramidetrihexoside antibodies. 4. A human anti-P 1 ,P,P k serum from a person with the rare phenotype p, which contains anticeramidetrihexoside (anti-P k ) activity was compared with the rabbit anti-ceramidetrihexoside serum. The two antisera showed very similar characteristics with regard to the anti-ceramidetrihexoside activity.

Effect of 2-deoxyglucose on lysosomal enzymes in cultured human skin fibroblasts☆

Abstract Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins, to the medium of confluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about 1 mM 2-deoxyglucose. The inhibition by 2-deoxyglucose is reversible. The K m , pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without 2-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95% of the β-hexosaminidase and α-galactosidase activity and about 65 % of the acid phosphatase activity with β-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of 2-deoxyglucose, the specific activity of alkaline phosphodiesterase I, a plasma membrane glycoprotein is lowered. 2-Deoxyglucose has no effect on the specific activity of succinate dehydrogenase, lactate dehydrogenase or total cellular protein.

Further characterization of two forms of N-acetyl-α-galactosaminidase from human liver

Abstract 1. 1. 2 form of N-acetyl-α-galactosaminidase (EC 3.2.1.22) can be isolated from human liver using Sepharose 4B-concanavalin A chromatography, followed by CM-cellulose chromatography. Both forms hydrolyse not only p-nitrophenyl-N-acetyl-α-galactosaminide but also p-nitrophenyl-α-galactoside. These two forms were formerly known as α-galactosidase B and A-like α-galactosidase (Schram, A.W., Hamers, M.N., Brouwer-Kelder, B., Donker-Koopman, W.E. and Tager, J.M. (1977) Biochim. Biophys. Acta 482, 125–137; Schram, A.W., Hamers, M.N. and Tager, J.M. (1977) Biochim. Biophys. Acta 482, 138–144). 2. 2. In fresh normal liver, most of the N-acetyl-α-galactosaminidase activity is found in the fractions containing α-galactosidase B and a minor portion in the fractions containing α-galactosidase A. However, the minor peak of N-acetyl-α-galactosaminidase activity does not coincide with the peak of α-galactosidase A activity. Both peaks of N-acetyl-α-galactosaminidase activity can be precipitated by antiserum raised against α-galactosidase B but not by antiserum raised against α-galactosidase A. 3. 3. In fresh Fabry liver (which contains no α-galactosidase A) more than 98% of the N-acetyl-α-galactosaminidase activity was recovered in the fractions containing α-galactosidase B. Less than 2% was found in the fractions containing A-like α-galactosidase. Upon aging of the liver, the yield of the latter form increased (to almost 25% after 17 months storage at −20°C). 4. 4. The conversion of N-acetyl-α-galactosaminidase to the second form upon aging was accompanied by a decrease in apparent molecular weight from 110 000 ± 5000 to 99 000 ± 3000, as measured by Sephacryl S-200 chromatography. 5. 5. The kinetic properties of the enzyme change slightly upon aging. In contrast, there is no effect of aging on the pH optimum of the enzyme. 6. 6. This is concluded that only one lysosomal N-acetyl-α-galactosaminidase is present in human liver and that one of the two forms of N-acetyl-α-galactosaminidase observed in extracts of liver is formed by chemical modification of the enzyme during storage of the liver or during storage of the purified preparation.

A new immunochemical method for the quantitative measurement of specific gene products in man-rodent somatic cell hybrids

SummaryAn immunochemical method has been developed for the quantitative determination of species-specific gene products, for instance a-galactosidase and N-acetyl-a-galactosaminidase, in man-rodent hybrid cells and in the parental cell lines. Antisera raised against the purified enzymes are covalently coupled to Sepharose 4B. The gene products are specifically removed from a cell lysate by incubating with the appropriate Sepharose-coupled antiserum. After centrifugation followed by washing of the precipitated Sepharose, the enzymic activity can be quantitatively measured on the Sepharose beads. With this technique it has been demonstrated that the ability of human N-acetyl-a-galactosaminidase (also known as a-galactosidase B) to hydrolyze a-galactosidic linkages is lost when the enzyme is expressed in man-Chinese hamster hybrid cells.

Properties of immobilized FIG α-galactosidase and effect of ceramide-3 content of plasma from patients with fabry's disease

The possibility of lowering the level of ceramide-3 (galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosyl-beta(1 leads to 1)-ceramide) in the plasma of patients with Fabrys disease was investigated. An immobilized alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was prepared by coupling purified fig alpha-galactosidase to Sepharose 4B. The pH optimum for the hydrolysis of the artificial substrate p-nitro-phenyl-alpha-D-galactopyranoside was shifted by approx. 0.5--1.0 pH unit to higher pH values upon coupling of the enzyme to Sepharose 4B. The immobilized enzyme was more stable than the native enzyme to incubation at 60 degrees C. The immobilized enzyme was able to hydrolyse ceramide-3 either at pH 4.5 or at pH 7.4 in an artificial system in which sodium taurocholate was used to solubilize the substrate. In contrast, when the immobilized enzyme was incubated with normal plasma or plasma from a patient with Fabrys disease, in which elevated levels of ceramide-3 occur, no hydrolysis of the glycosphingo-lipid could be detected. The results suggest that lowering of level of ceramide-3 in plasma from patients with Fabrys disease by enzymic means is not feasible.

Factors affecting the hydrolysis of ceramide-3 by α-galactosidase a from human liver

Abstract 1. 1. The effect of detergents on the catalytic properties of α-galactosidase from human liver was studied using p- nitrophenyl-α-galactosidase and galactosyl -α(1→4)- galactosyl -β(1→4)- glucosylceramide (ceramide-3) as substrates. 2. 2. The hydrolysis of p- nitrophenyl-α-galactoside by α-galactosidase was inhibited by commercial preparations of sodium taurocholate and by taurocholate purified from these preparations by thin-layer chromatography. The extent of inhibition was dependent on the concentration of the detergent and on the amount of protein present. The impurities present in the preparation also inhibited the hydrolysis. 3. 3. The inhibition of taurocholate preparations of p- nitrophenyl-α-galactoside hydrolysis was pH-dependent. 4. 4. The inhibition by taurocholate of p- nitrophenyl-α-galactoside hydrolysis can be partly overcome by adding glycosphingolipids. 5. 5. No significant hydrolysis of ceramide-3 occurs in the absense of detergent. Upon adding increasing concentrations of taurocholate, the rate of hydrolysis increases to a maximum value. At still higher taurocholate concentrations the activity decreases. 6. 6. The concentrations of taurocholate giving a maximal rate of hydrolysis of ceramide-3 is dependent on the amount of protein present and independent of the ceramide-3 concentration. 7. 7. When the pH dependence of the rate of hydrolysis of ceramide-3 was measured in the presence of a commercially available preparation of pure taurocholate or of crude taurocholate, curves with different shapes were obtained