Lactose metabolism. IV. The function of uridine triphosphate in the interconversion of galactose-1-phosphate and glucose-1-phosphate.
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Syntheses of methyl 6-(alpha-D-galactopyranosyl phosphate)- and p-nitrophenyl 3-(alpha-D-galactopyranosyl phosphate)-alpha-D-galactopyranosides were performed through coupling of 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl hydrogenphosphonate with the partially acylated galactopyranosides, in the presence of trimethylacetyl chloride, followed by oxidation and debenzoylation. The data of 1H, 13C, and 31P NMR spectra of the synthesized phosphate diesters are reported.
Galactose-1-phosphate uridylytransferase (E.C.2.7.12) activity was measured in both lymphoid and erythroid cells from patients with chronic lymphocytic leukemia (CLL). Decreased enzyme activity was found in both cell types using two assay methods. The results suggest the presence of an inhibitor of the enzyme in CLL patients. A correlation between decreased uridyl transferase activity and glycogen accumulation in CLL is postulated.
34 persons with G-6-PD deficiency were diagnosed, and the pathological enzyme-variants of red blood cells were characterized according to the recommendations of WHO. We conclude from the differing residual G-6-PD-activities in red blood cells of the propositi and the differing reactivity of the enzyme in kinetic and physicochemical characterizations that a multiple variety of rare pathological G-6-PD variants exists in the GDR. Using the estimated enzymeparameters it was not possible in all cases to compare directly the newly demonstrated G-6-PD variants with cases already described in the literature. In addition, the differing combinations of parameters render a classification more difficult.
Porcine submaxillary beta-galactoside alpha(1----2)-fucosyltransferase is known to transfer a fucosyl residue from guanosine 5'-diphosphofucose (GDP-fucose) to the 2-OH group of beta-D-galactopyranosides with inversion of configuration at the fucopyranosyl anomeric carbon. A bisubstrate analog (1) of the postulated transition-state for this reaction, which has O-2 of phenyl beta-D-galactopyranoside attached to the terminal phosphorous of GDP through a flexible ethylene bridge, has been chemically synthesized and evaluated as an inhibitor of this enzyme. Compound 1 was found to be a competitive inhibitor with respect to both GDP-fucose and phenyl beta-D-galactopyranoside for both the membrane-bound and soluble forms of the fucosyltransferase. It was also a competitive inhibitor with respect to the alternate acceptor beta DGal(1----3)beta DGlcNAcO(CH2)8-COOMe. The Ki values were in the range 2.3-16 microM. Compound 1 is the first example of a bisubstrate analog inhibitor for a glycosyltransferase which binds to both the acceptor and donor recognition sites of the enzyme. The potential of a bisubstrate analog strategy for the production of specific glycosyltransferase inhibitors is discussed.
Clinical and biochemical diagnostic studies concerned 17 cases of galactosemia coming from 15 not consauguineous families. Galactosemia was diagnosed between 1-st day and 11-th month of life. Tentative diagnosis based on clinical picture was made in 12 infants, others were detected through family history of galactosemia and/or biochemical newborn screening carried out at the National Research Institute of Mother and Child since 1969. Clinical symptoms of galactosemia occurred in most patients in the first week of life. They were the following (tab. II): hepatomegaly (in 94%), jaundice (81%), splenomegaly (79%), vomitus (62%) and diarrhoea in 56% of patients. Cataract was found in 6 infants (38%). Biochemical diagnosis was based on the results of enzymatic estimation of galactose-1-phosphate uridyl transferase activity in blood, galactose-1-phosphate in red blood cells and galactose in blood and urine. No activity of galactose-1-phosphate uridyl transferase was found in all patients, and the concentration of galactose-1-phosphate was higher than 25 mg/100 ml of red blood cells. High galactose level was observed in blood and urine in all patients with typical clinical course of galactosemia. In 2 patients however without clinical symptoms of the disease only trace amounts of galactose was detected in blood and urine. All these patients were treated with galactose free diet.
In July 1983, the Manitoba Perinatal Screening Programme modified its existing procedure for neonatal screening for galactosemia by introducing quantitation of total galactose plus galactose-1-phosphate from dried blood spots using the Multistat centrifugal analyzer. The first 4 years of experience with this method in combination with the Beutler spot test for galactose-1-phosphate uridyl transferase activity is the subject of this report. Of 70,336 newborns screened, 142 (0.20%) met the criteria for clinical follow up. Of these, one child was confirmed to have classical galactosemia and nine children were found to be Duarte/galactosemia genetic compounds. This method of galactosemia screening has proven to be rapid, sensitive, efficient, and the method of choice for mass screening of disorders of galactose metabolism.
We describe a microassay for measuring galactose (Gal) and galactose 1-phosphate (Gal-1-P) in dried blood spots. After a coupled enzyme reaction involving galactose dehydrogenase (GADH, EC 1.1.1.48) and alkaline phosphatase (AP, EC 3.1.3.1) in a microplate well, NADH fluorescence is measured by a highly sensitive fluorometric microplate reader, capable of rapid measurement of fluorescence (2 min per 96 samples). Within- and between-run CVs for measurements of Gal at 90 mg/L with Gal-1-P at 130 mg/L were both less than 5% (n = 8), and analytical recoveries for Gal at 90 mg/L and Gal-1-P at 130 mg/L were 98% and 92%, respectively. Five hundred dried blood-spot samples can be assayed within 2 h, with full calculation of results by an on-line microcomputer. This rapid and reliable assay system is very useful for the routine screening of newborns for galactosemia.
The increase of intracellular cyclic AMP levels suppresses the cytotoxic activities of lymphocytes and monosaccharides interfere with cell to cell and cell to cytokine interactions, and these effects on natural killer (NK)/antibody-dependent cell-mediated cytotoxicity (ADCC) and lymphokine activated killer (LAK) activities were examined. Dibutyryl cyclic AMP markedly suppressed NK, IL-2-augmented NK and ADCC activities in a dose-dependent manner but not previously induced LAK activity. Induction of LAK was inhibited. Dibutyryl cyclic GMP had no effect. Addition of mannose 6-phosphate and galactose 6-phosphate strongly inhibited NK and ADCC activities, but not LAK activity. These results suggest that the lytic mechanism for NK and ADCC activity is different from that of LAK activity.
alpha-Lactalbumin was isolated from milk of M. eugenii and its concentration in milk samples taken at various times during lactation (0-40 weeks post partum) was determined by single radial immunodiffusion using rabbit antiserum to the purified protein. The alpha-lactalbumin concentration remained almost constant throughout lactation even though the concentration of total lactose (free lactose plus lactose contained in oligosaccharides) fell to zero after 34 weeks post partum. This fall in lactose was accompanied by a rise in the free galactose and glucose concentrations and marked increases in UDP-galactose hydrolase, nucleotide pyrophosphatase, alkaline phosphatase and acid beta-galactosidase activities. It is suggested that the in vitro hydrolysis of UDP-galactose was due to nucleotide pyrophosphatase and that this enzyme may also play a role in vivo late in lactation by making UDP-galactose unavailable for the synthesis of lactose. Alternatively, lactose and lactose-containing oligosaccharides might be degraded by the acid beta-galactosidase during or after secretion.
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