Glycosylation of endogenous proteins through dolichol derivatives in reticulocyte plasma membranes.

It has been found that rabbit reticulocyte plasma membranes contain the enzymatic activities responsible for the synthesis of all the polyprenol-bound sugars that are intermediates in the glycosylation of proteins. The final reaction in which an oligosaccharide in transferred from a dolichol pyrophosphate derivative to an endogenous protein was detected in reticulocyte but not in erythrocyte plasma membranes.

N-acetyl-beta-D-hexosaminidase A from rat urine: partial purification and characterization.

N-Acetyl-beta-D-hexosaminidase A was purified from rat urine by ion-exchange chromatography on DEAE-cellulose, followed by concanavalin A chromatography, and finally by chromatography on 2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta-glucosylamine-Se pharose 4B. The enzyme was purified 482-fold with a yield of about 7%. The optimal pH was 4.5 for N-acetyl-glucosaminidase activity and 4.0-4.5 for N-acetylgalactosaminidase activity. The enzyme was heat-labile and stable from pH 4.5 to pH 7.0 but it was very unstable at lower pH values. Km values were 0.55 mM and 0.059 mM, respectively. The glycoprotein nature of the enzyme was deduced from its behavior on concanavalin A. The effect of some carbohydrates and ionic compounds on the activities of the enzyme was studied. When N-acetyl-D-glucosaminolactone and N-acetyl-D-galactosaminolactone were used as inhibitors, Ki values were also calculated.

Synthesis of dolichol derivatives in human erythrocyte membranes.

Human erythrocyte membranes contain the enzymes responsible for the synthesis of dolichol-P-glucose, dolichol-P-mannose, dolichol-PP-N-acetylglucosamine, dolichol-PP-NN' diacetylchitobiose and of dolichol-PP-oligosaccharides containing NN' diacetylchitobiose and mannose or the same sugar residues plus glucose. The transfer of the oligosaccharide moieties from the dolichol-PP-oligosaccharides to endogenous proteins could not be detected. These enzymes appeared to be integral membrane proteins.

Maternal-fetal relationship in ammonia metabolism during late gestation in the rat.

The concentrations of the major metabolites for nitrogen excretion and/or transport in maternal and fetal blood and amniotic fluid during the last 2 days of gestation were investigated. Alanine, glutamine, ammonia and allantoin accumulated in amniotic fluid during late gestation. Urea concentrations increased in amniotic fluid though only during the last day of gestation, suggesting that urea is taken up by the mother through the amniotic membranes. Glutamate did not accumulate in amniotic fluid during late gestation although high concentrations of it were found in fetal blood in the same circumstances, suggesting the occurrence of a mechanism for preventing fetal glutamate disposal.

Separation and properties of a "neutral" hexosaminidase from embryonic chicken brain.

1. A "neutral" hexosaminidase has been separated from other hexosaminidase forms (I and II) by DEAE-cellulose chromatography and characterized in embryonic (16-days old) and 1-day old chicken brains. 2. Its properties differ from those of the forms I and II. It has optimum activity at about pH 6.0 and can be eluted from DEAE-cellulose with 0.25 M KCl only. 3. It has no N-acetylgalactosaminidase activity and cannot be successfully detected after isoelectric focusing since it is very acidic and completely unstable below pH 5.0. 4. "Neutral" hexosaminidase is heat-stable at pH 6.0 and is inhibited by chloride. 5. These properties, very different from those of forms I and II, suggest that this "neutral" form of hexosaminidase would be very similar to known hexosaminidase C separated from other materials. 6. We have found no significant differences for the above-mentioned three forms in chick embryos (16-days old) in comparison with those from 1-day old chicken.

Glycoprotein assembly in Leishmania mexicana.

The oligosaccharide transferred from a dolichol-P-P derivative to proteins in the assembly of N-linked glycoproteins in Leishmania mexicana appeared to be Man6GlcNAc2. It was found that this compound underwent transient glucosylation once bound to protein but that Man6GlcNAc2 was the oligosaccharide present in mature glycoproteins. No complex type saccharides were detected. The structure of the oligosaccharide appeared to be similar to that of the core of compounds transferred from dolichol-P-P derivatives in protein glycosylation in Trypanosoma cruzi or animal cells.

Ammonia metabolism during the suckling period in the rat.

1. The time-courses of blood glutamine, glutamate, alanine, ammonia, urea and allantoin concentrations during the first 15 days of extrauterine life were studied. 2. Glutamine and glutamate concentrations followed the same pattern and correlated positively, suggesting that both amino acids are utilized or released synchronously. 3. Alanine concentrations decreased during the first 3 days, reaching levels close to those of adults which persisted up to the end of the observation period. 4. A highly significant correlation was found between ammonia and urea concentrations, suggesting that during the suckling period urea synthesis may be limited by blood ammonia availability. 5. The time-course of allantoin concentrations suggests that the synthesis of purines was enhanced during the first day, decreasing sharply during the 2nd to steeply increase to the end of the suckling period.

alpha-D-mannosidase forms in chicken liver.

Two forms (I and II) of alpha-D-mannosidase have been separated by ion-exchange chromatography on DEAE-cellulose from embryonic chicken liver. A third form (III), which is absent in embryos, was also separated from 4-day-old chickens. The optimum pH of form I is at pH 5.0. Form II is named "neutral" because it shows maximal activity at pH 6.5. The optimum pH of form III is 4.5. Forms I and III are heat-stable at 50 degrees C for 1 hr, whereas form II is very unstable under these conditions. Zn2+ and Mg2+ have been found to increase the alpha-D-mannosidase activity of forms I and II. In contrast, Co2+ increases mannosidase I activity and inhibits form II from 18-day-old embryos. alpha-Methyl-D-mannoside, N-acetyl-D-mannosamine and D-mannosamine were found to be inhibitors of both forms I and II. "Neutral" mannosidase was also inhibited by chloride. Competitive inhibition by D-mannose was also studied and Ki values are given.

Kinetic studies of four enzyme forms of beta-N-acetylhexosaminidase from embryonic chicken brain.

The separation of four different hexosaminidase forms from embryonic chicken brain (16-day-old) has been performed by ion-exchange chromatography. Two different DEAE-cellulose columns have been used: a first one at pH 7.2 and a second one at pH 6.0. Km and Vmax values were estimated from the Lineweaver-Burk or Dixon plots and ki from the Dixon plots, using N-acetyl-D-glucosamine or N-acetyl-D-galactosamine as inhibitors. In both cases we found a kind of competitive inhibition in which Lineweaver-Burk and Dixon plots curve downwards.

Separation and characterization of four forms of beta-N-acetylhexosaminidase from chicken brain.

Chicken brain beta-N-acetylhexosaminidases from embryos (16 and 21 days old), newborns (1 and 4 days old), and adults (3 1/2 months and 2 years old) were separated into four different forms by ion exchange chromatography on diethylaminoethyl-cellulose. Three of these forms were "acid" hexosaminidases (I, IIA, and IIB), and the fourth was a "neutral" form. Throughout development of the chicken, forms IIA and III maintained the same activity ratio, whereas that for form I decreased and that for form IIB showed an increase.

Metabolic fuel utilization and pyruvate oxidation during the postnatal period.

The transplacental supply of nutrients is interrupted at birth, which diverts maternal metabolism to lactation. After birth, energy homeostasis is rapidly regained through milk nutrients which supply the newborn with the fatty acids and ketone bodies required for neonatal development. However, immediately after birth and before the onset of suckling there is a time lapse in which the newborn undergoes a unique kind of starvation. During this period glucose is scarce and ketone bodies are not available owing to the delay in ketogenesis. Under these circumstances, the newborn is supplied with another metabolic fuel, lactate, which is utilized as a source of energy and carbon skeletons. Neonatal rat lung, heart, liver and brain utilize lactate for energy production and lipogenesis. Lactate is also utilized by the brain of human babies with type I glycogenosis. Both rat neurons and astrocytes in primary culture actively use lactate as an oxidizable substrate and as a precursor of phospholipids and sterols. Lactate oxidation is enhanced by dichloroacetate, an inhibitor of the pyruvate dehydrogenase kinase in neurons but not in astrocytes, suggesting that the pyruvate dehydrogenase is regulated differently in each type of cell. Despite the low activity of this enzyme in newborn brain, pyruvate decarboxylation is the main fate of glucose in both neurons and astrocytes. The occurrence of a yeast-like pyruvate decarboxylase activity in neonatal brain may explain these results.

Effect of tobramycin and gentamicin on the activity of some glycosidases in rat serum and urine.

beta-Galactosidase, alpha-D-mannosidase, alpha-L-fucosidase and N-acetyl-beta-D-glucosaminidase activities were assayed in serum and urine from rats treated with three different doses of the nephrotoxic antibiotic tobramycin (100 mg/kg/day for 5 days, 10 mg/kg/day for 10 days and 5 mg/kg/day for 20 days) and gentamicin (100 mg/kg/day for 5 days). A significant increase of beta-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-L-fucosidase activities occurred in urine following the administration of high doses of antibiotic. The enzyme activity was dependent on the dose level used. The excretion of alpha-D-mannosidase was atypical and elevated activities were observed on some days but no pattern of excretion of this enzyme was established. No change in any of the four glycosidase activities was found in serum of treated rats. The results obtained when high doses of gentamicin were employed are similar to those obtained with a similar dose of tobramycin. These results indicate that the assay of urinary glycosidase activities provides a useful method for monitoring the nephrotoxicity of antibiotics.

Evidence that transient glucosylation of protein-linked Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 occurs in rat liver and Phaseolus vulgaris cells.

Formation of protein-linked Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 was detected in rat liver slices and Phaseolus vulgaris seeds incubated with [U-14C]glucose. Similar compounds were not synthesized in Saccharomyces cerevisiae cells incubated under similar conditions. Rat liver microsomes were incubated with [glucose-U-14C] Glc3Man9GlcNAc2-P-P-dolichol or UDP-[U-14C]Glc as glycosyl donors. Only in the latter condition protein-linked Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 were formed. Addition of mannooligosaccharides that strongly inhibited alpha 1-2-mannosidases to incubation mixtures containing rat liver microsomes and UDP-[U-14C]Glc did not prevent formation of protein-bound Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 . Furthermore, the presence of amphomycin in reaction mixtures containing liver membranes and UDP-[U-14C]Glc completely abolished synthesis of glucosylated derivatives of dolichol without affecting formation of protein-linked Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 . The results reported above indicated that under the experimental conditions employed protein-bound Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 were formed by glucosylation of unglucosylated oligosaccharides. Results obtained in pulse-chase experiments performed in vitro also supported this conclusion. UDP-Glc appeared to be the donor of the glucosyl residues. The rough endoplasmic reticulum was found to be the main subcellular site of protein glucosylation. It is tentatively suggested that this process could prevent extensive degradation of oligosaccharides by mannosidases during transit of glycoproteins through the endoplasmic reticulum.

Effect of postnatal hypoxia on ammonia metabolism during the early neonatal period in the rat.

The effect of experimental hypoxia on blood glutamine, glutamate, urea, ammonia, allantoin, hypoxanthine, xanthine, urate, orotate and lactate concentrations and on PO2, PCO2 and pH in term delivered newborn rats during the first 4 h after delivery were studied. Hypoxia increased blood glutamine, glutamate, allantoin and 'xanthines' (hypoxanthine + xanthine + uric acid) concentrations but decreased blood urea and ammonia concentrations. These results suggest that hypoxia inhibited ureogenesis by decreasing the ammonia available for urea synthesis.