Treatment of cystinosis with cysteamine. A pilot study determining dose and form of application.

A pilot study with cysteamine treatment was performed in three children with the nephropathic form of cystinosis. Two children underwent renal transplantation shortly before treatment. The aim of the study was to find a practicable form of application and a corresponding effective dose. Cysteamine in gelatine capsules together with 0.2% silicic acid as a dessicator turned out to be the most acceptable galenic form, compared to sirup or suppositories. Among three dosage regimens, the dosage of 50 mg/kg/day is effective as judged by the leucocyte cystine content, even if given in only three doses per day. No side effects of the cysteamine treatment (even at a dose of 90 mg/kg/day) were noted. Whether this treatment is preventing progression of disease will have to be examined either in transplanted patients by measuring non-renal parameters or in very young infants with cystinosis whose kidneys are not damaged yet.

The role of enzyme variants, polymorphisms and enzyme hybrids in enzyme deficiency conditions.

Based on the heterogeneity observed in two red cell enzymes, i.e. glucose 6-phosphate dehydrogenase (E.C. 1.1.1.49) and catalase (E.C.1.11.1.6), the role of different types of enzyme variants in enzyme deficiency conditions is discussed. For theoretical and practical reasons variants of unusually low specific activity and of low stability have to be distinguished. Whereas in the former type the activity level in blood more or less reflects the situation in other tissues, this is not the case for unstable mutants, e.g. the enzyme variant found in Swiss-type acatalasemia. In heterozygous carriers the situation can be complicated by the fact that variants of oligomer enzymes (e.g. catalase) are present as molecular hybrids exerting almost normal stability and activity.

The microheterogeneity of human plasma alpha1-acid glycoprotein.

alpha1-Acid glycoprotein was isolated in the homogeneous state from the plasma of 33 normal individuals and subjected to analytical isoelectric focusing before and after treatment with neuraminidase. The native glycoprotein preparations, resolved into 6 to 8 bands, were quantitated and grouped into two classes according to the patterns obtained: One class exhibited a relatively anodic and the other a relatively cathodic distribution of the protein bands. The isoelectric points of these bands ranged from pH 2.90 to 3.30. After treatment with neuraminidase the resulting asialo-glycoproteins were also quantitated and afforded two types of fundamentally different patterns from those mentioned above, namely one type with one and the other type with two main bands and both exhibiting several minor components. The isoelectric points of the main bands were found to be of pH 4.55 and 4.70 while those of the minor bands were at both the anodic and cathodic side of the major bands. No apparent relationship between the patterns of the native and those of the asialo-glycoproteins could be established. In addition, a new variant was noted whose major band focused at a pH of 5.0. The microheterogeneity of alpha1-acid glycoprotein is thus interpreted to be due to the amino acid replacements of this protein in combination with the linkages of the sialyl to the galactosyl residues in the native protein.

Protection of human neutrophils by endogenous catalase: studies with cells from catalase-deficient individuals.

To investigate the importance of catalase as a protecting enzyme against oxidative damage in phagocytic leukocytes, we have tested the functional capacity of neutrophils from two individuals homozygous for Swiss-type acatalasemia and from two individuals heterozygous for this deficiency. In the former cells, 25-30% of residual activity of catalase was present. In the latter cells, the values were close to normal. Chemotaxis towards casein, release of lysosomal enzymes and hydrogen peroxide during phagocytosis of zymosan, and intracellular killing of Staphylococcus aureus were normal in all cells tested. Inhibition of heme enzymes with azide (2 mM) enhanced the respiration and hexose monophosphate shunt activity of normal, but not of homozygous acatalasemic, neutrophils. This indicates that the enhancement in normal cells is, at least in part, due to catalase inhibition. After 15 min preincubation with an H(2)O(2)-generating system (glucose plus glucose oxidase), the respiratory response to zymosan phagocytosis was strongly depressed in the homozygous acatalasemic and in normal, azide-treated neutrophils, but not in normal, untreated cells. Under these conditions, the release of lysosomal enzymes was depressed and that of lactate dehydrogenase enhanced, in catalase-deficient and in catalase-inhibited, but not in normal, neutrophils. During prolonged incubation with the H(2)O(2)-generating system (30-60 min), the reduction level of intracellular glutathione remained high and the hexose monophosphate shunt continued to operate normally in all cells tested. Thus, although the function of neutrophils without catalase activity was depressed by extracellular hydrogen peroxide, the H(2)O(2) degradation via the glutathione redox system remained operative. The results indicate that the glutathione redox system by itself efficiently protects phagocytosing neutrophils against their own oxidative products. During heavy external oxidative stress, however, both catalase and the glutathione redox system are needed for adequate protection.

Catalase and dehydroascorbate reductase in human polymorphonuclear leukocytes (PMN): possible functional relationship.

In leukocytes (PMN) of individuals with Swiss type acatalasemia, the rate of dehydroascorbate reduction is 4 times normal. This observation suggests that the protective function served by catalase in human PMN is supported by dehydroascorbate reductase.

The charge heterogeneity of soluble human galactosyltransferases isolated from milk, amniotic fluid and malignant ascites.

UDP-galactose: N-acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N-acetylglucosamine--agarose and alpha-lactalbumin--agarose yielded an enzyme preparation homogeneous by size. Under non-denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved faster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N-acetylglucosamine, ovalbumin, sialic-acid-free ovine submaxillary mucin and glucose, in the presence of alpha-lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed.

Properties of human erythrocyte catalases after crosslinking with bifunctional reagents. Symmetry of the quaternary structure.

Normal erythrocyte catalase, the enzyme present in the blood of Swiss acatalasemic heterozygotes, and their hybrid produced in vitro, were studied after crosslinking with bifunctional reagents. On theoretical grounds [cf. Hajdu, J., Bartha, F. & Friedrich, P. (1976) Eur. J. Biochem. 68, 373--383] it is inferred from the dodecylsulphate gel electrophoretic patterns obtained after treating catalase with diimidates of various chain lengths that the enzyme is an isologous tetramer (D2 symmetry). The minimal distances between crosslinkable primary amino groups across the three domains of bonding are different. Reaction with diimidates causes a moderate loss of enzyme activity in all three enzyme types due to amidination rather than crosslink formation. On the other hand, crosslinking stabilizes the enzyme against urea and heat inactivation. This is most prominent with heterozygote acatalasemic catalase. Crosslinking markedly prevents the development of peroxidase activity that can be elicited in catalases by urea treatment. The role of the quaternary structure of the protein in the relationship between catalase and peroxidase activities is discussed.

Unstable mutants and molecular hybrids in enzyme deficiency conditions.

Multiple molecular forms contribute to various types of enzyme heterogeneity: "Iso(en)-zymes" and "Allozymes" (enzyme variants) are of genetic origin whereas "Metazymes" represent secondary modifications of epigenetic nature. The concept of variability originates from the discovery of a large number of enzyme variants. Structural gene mutations can lead to enzyme variants of low specific activity or reduced stability and can cause enzyme deficiencies. In heterozygous carriers of oligomeric enzyme defects, the formation of hybrid molecules is possible by random assembly of simultaneously synthetized normal and mutant subunits. The study of normal processes as well as enzyme anomalies contributes essentially to better understanding of biochemical individuality and evolutionary events at a molecular level.

Properties of erythrocyte catalase from homozygotes and heterozygotes for Swiss-type acatalasemia.

The unstable catalase variant found in the blood of individuals homozygous for Swiss-type acatalasemia and the enzyme species present in heterozygous carriers of this rare defect have been further characterized. The mutant enzyme isolated from acatalasemic red cells is considerably more heat labile and differs in electrophoretic mobility from the normal enzyme. Catalase preparations obtained from heterozygotes consist of an apparently uniform enzyme species, probably representing a molecular hybrid, with properties intermediate to those of the normal and the variant enzyme. However, antigenic identity of catalase from all three sources is observed. Model experiments indicate that hybrid catalase molecules can be produced by recombining normal and variant dimer subunits. Fractionation of erythrocytes according to density and age shows that most of the residual catalase activity is localized in juvenile acatalasemic cells, whereas in normal and heterozygous individuals the catalase activity level does not alter significantly during the life span of the red cells. These findings agree with the observation that there is no gene dosage in heterozygotes, their catalase activity values falling within the normal range.

Properties of leukocyte catalase in Swiss type acatalasemia: a comparative study of normals, heterozygotes and homozygotes.

Properties of leukocyte catalase from individuals heterozygous and homozygous for Swiss type acatalasemia were found to differ from those of the normal enzyme as well as interindividually in regard to heat stability and electrophoretic mobility. Molecular hybridization is discussed as a possible explanation for the presence of intermediate catalase species. Antigenic identity of catalase from all sources is confirmed.