Normal human erythrocyte catalase, when isolated by the method of Mörikofer et al. [Eur. J. Biochem. 11 (1969) 49–57], is heterogenous in two respects: (a) there are alternative molecular forms; (b) besides the active tetramer, enzyme dissociation products (dimer, monomer) are present. By means of exclusion chromatography on Sephadex G‐150 it was shown that catalase preparations contain dimer and monomer in small amounts (∼ 4%). Upon storage, the yield in subunits can be considerably enhanced (up to 15%). Residual catalase activity in the blood of an individual homozygous for Swiss Type Acatalasemia (1–2% of normal level) consists of a catalase variant, which is unstable, but of approximately normal specific activity. In this mutant, the tendency of dissociation into subunits is greatly enhanced. Upon purification, little tetramer but appreciable amounts of the inactive dimer are obtained. The antigenic properties of these molecular species were investigated using anti‐catalase and anti‐catalase‐subunit immunoglobulins G. There is complete antigenic identity between the normal and the variant catalase; the same is true for the normal and variant dimer. Different antigenic properties of dimer and monomer, however, offer evidence for the existence of hidden determinants in the subunits.

Human erythrocytes were separated by buoyant density ultracentrifugation into fractions of progressively increasing mean cell age to measure the changes in glycolytic activity that occur during their 120-day life-span. The maximal activities of all glycolytic enzymes were shown to decline exponentially with cell age. Only three glycolytic enzymes exhibited a marked rate of decline with a tl/2 shorter than the cell life-span: hexokinase, aldolase, and pyruvate kinase. ganic phosphate), showed a fourfold decrease through the erythrocyte lifespan; lactate production also declined, but at a slower rate. When incubating conditions were altered by the introduction of a metabolic stimulus (either high phosphate for glycolysis, or methylene blue for the pentose pathway) the youngest cell fractions responded with decidedly increased rates of glucose consumption and lactate production. However, this ability gradually declined with cell aging, and ultimately, the oldest cells had metabolic rates as low as if there were no stimulus present, The oldest erythrocytes appear to have lost the flexibility needed to respond t o metabolic stress and are more vulnerable to events in the circulation that may require the ability to increase the basal rate. This defect is probably responsible for the disappearance of aged erythrocytes from the circulation.Glucose utilization, when measured in steady-state conditions (1 mM inor-

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 a-lactalbuniin -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 a-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.Biosynthesis of the heteropolysaccharide moieties of complex carbohydrates is catalyzed by glycosyltransferases (for review see [l]). The information on their structure is scarce. One of the best known glycosyltransferases is UDP-galactose : N-acetylglucosamine galactosyltransferase or A protein of the lactose synthetase complex (for review see [ 2 ] ) . This enzyme has been purified from various animal and human body fluids [3-81 but, with the exception of amino acid and carbohydrate content of the bovine milk enzyme [9], no additional information on the structure is available. Preliminary work indicated charge heterogeneity of galactosyltransferase as revealed by activity measurements of crude enzyme preparations which were subjected to isoelectric focusing [lo]. The present investigation was carried out in order to characterize the charge heterogeneity of pure galactosyltransferase. Since reports dealing with electrophoretic variants associated with cancer have recently been published [11--131, it seemed of interest to correlate the patterns obtained for the Abbreviations. Asialo-mucin, sialic-acid-free ovine submaxillary mucin; butyl-PBD, 2-(~-tert-butylphenyl)-5-(4-bisphenylyl)-1,3,4-oxadiazole.Enzynw. UDP-galactose :N-acetylglucosamine galactosyltransferase (EC 2.4.1.22). milk enzyme to the enzymes isolated from other sources, such as malignant ascites and amniotic fluid. The latter was chosen in order to investigate the possible occurrence of an onco-fetal variant in ascites (for review see [14]). The present work describes a higher electrophoretic mobility of galactosyltransferases from ascites and amniotic fluid under nondenaturing conditions when compared to the milk enzyme. This difference was due solely to ...