Reaction of human UMP-CMP kinase with natural and analog substrates.

UMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site.     

I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates

Cultured skin fibroblasts from a patient with I-Cell disease (mucolipidosis II) were assayed for a number of lysosomal enzymes using both natural and synthetic substrates. The cells from this patient were found to have very low activity for galactosylceramide β-galactosidase, lactosylceramide β-galactosidases (using two assay methods that measure different enzymes), G_M1 ganglioside β-galactosidase and sphingomyelinase. Glucosylceramide β-glucosidase activity was found to be normal. Acid hydrolase activities toward many synthetic substrate were measured and all except β-glucosidase and acid phosphatase were found to be extremely low (as has been reported by others). Acid phosphatase and β-glucosidase were in the low normal range. These studies expand on previously published reports on I-Cell disease that only present data from synthetic substrates, and also report the fibroblast culture deficiencies of galactosyl-ceramide β-galactosidase (the Krabbe disease enzyme) and sphingomyelinase (the Niemann-Pick disease enzyme) activities for the first time. Those two enzymes do not have a readily available synthetic analog to assay. Acid β-galactosidase activity measured with both the 4-methylumbelliferyl derivative and G_M1 ganglioside was partially deficient in leukocytes prepared from this patient. New methods for measuring 4-methylumbelliferyl-β-D-glucoside and glucosylceramide β-glucosidase activities are also presented.     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

Evaluation of cyclohexenoesculetin-beta-D-galactoside and 8-hydroxyquinoline-beta-D-galactoside as substrates for the detection of beta-galactosidase.

We describe the synthesis of two new substrates for the detection of beta-galactosidase and evaluate their performance in comparison with that of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). Of 171 Enterobacteriaceae strains that were able to hydrolyze X-Gal, 166 (97.1%) also hydrolyzed cyclohexenoesculetin-beta-D-galactoside whereas only 96 (56.1%) showed evidence of hydrolysis of 8-hydroxyquinoline-beta-D-galactoside. No false-positive results were observed with either substrate.     

P blood group regulation of glycosphingolipid levels in human erythrocytes.

The neutral glycosphingolipid content of normal human erythrocytes was analyzed by a new method which utilizes high performance liquid chromatography. This rapid and accurate technique permits the quantitation of each of the major neutral glycolipids from individual blood samples. A correlation between the P blood group and the relative quantities of neutral glycosphingolipids is demonstrated. Erythrocytes from P1 individuals are shown to contain more globotriaosylceramide and less lactosylceramide than do erythrocytes from P2 individuals. The results of these experiments suggest the existence of a new phenotype in the P blood group system, and have further implications regarding the biosynthesis of the P blood group glycosphingolipids.     

UDP-glucosyltransferase activity toward exogenous substrates in Drosophila melanogaster.

To investigate the capacity of Drosophila extracts to glucosylate exogenous substrates we have developed a fast and sensitive method for the detection of UDP-glucosyltransferase activity using 4-nitrophenol, 1-naphthol, or 2-naphthol as substrates. High-performance liquid chromatography was used to separate and quantitate the reaction products, allowing detection of activities that produced as little as 1 pmol of 2-naphthol glucoside (fluorescence detection) or 16 pmol of 4-nitrophenol glucoside (absorbance detection). Optimal activity was found at 43 degrees C and alkaline pH. The affinity of the Drosophila enzyme was 250-fold higher for 1-naphthol or 2-naphthol (Km approximately 4 microM) than for 4-nitrophenol and UDP-glucose (Km approximately 1 mM).     

Structure of the human erythrocyte blood group P1 glycosphingolipid.

A glycosphingolipid with blood group P1 activity was extracted from an acetone powder of human erythrocyte stroma with chloroform-methanol. It was purified by chromatography on columns of silicic acid and by preparative thin-layer chromatography of the fully acetylated and deacetylated glycolipid. The purified glycolipid contained galactose, N-acetylglucosamine, and glucose in a molar ratio of 3:1:1. Treatment of the P1 glycolipid with fig alpha-galactosidase released a single galactosyl residue and destroyed the blood group activity, and the alpha-galactosidase product had the same chromatographic mobility as paragloboside. Substitution sites on the neutral sugars of the P1 glycolipid and the alpha-galactosidase product were established by identification of methylated alditol acetates, and substitution on N-acetylglucosamine was determined by identification of methyl glycoside derivatives. The terminal nonreducing disaccharide of the P1 glycolipid is Gal(alpha, 1 leads to 4)Gal. N-Acetylglucosamine was identified as the next sugar in sequence by mass spectrometric analysis of the permethylated P1 glycolipid. On the assumption that the glucose residue is linked to ceramide, we propose the following structure for the P1 glycolipid: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-NAc(beta, 1 leads to 4)Glc-Cer.     

Specificity of natural and artificial substrates for human Cdc25A

Cdc25A is a dual-specific protein phosphatase involved in the regulation of the kinase activity of Cdk-cyclin complexes in the eukaryotic cell cycle. To understand the mechanism of this important regulator, we have generated highly purified biochemical reagents to determine the kinetic constants for human Cdc25A with respect to a set of peptidic, artificial, and natural substrates. Cdc25A and its catalytic domain (dN25A) demonstrate very similar kinetics toward the artificial substrates p-nitrophenyl phosphate (k(cat)/K(m) = 15-25 M(-1) s(-1)) and 3-O-methylfluorescein phosphate (k(cat)/K(m) = 1.1-1.3 x 10(4) M(-1) s(-1)). Phospho-peptide substrates exhibit extremely low second-order rate constants and a flat specificity profile toward Cdc25A and dN25A (k(cat)/K(m) = 1 to 10 M(-1) s(-1)). In contrast to peptidic substrates, Cdc25A and dN25A are highly active phosphatases toward the natural substrate, T14- and Y15-bis-phosphorylated Cdk2/CycA complex (Cdk2-pTpY/CycA) with k(cat)/K(m) values of 1.0-1.1 x 10(6) M(-1) s(-1). In the context of the Cdk2-pTpY/CycA complex, phospho-threonine is preferred over phospho-tyrosine by more than 10-fold. The highly homologous catalytic domain of Cdc25c is essentially inactive toward Cdk2-pTpY/CycA. Taken together these data indicate that a significant degree of the specificity of Cdc25 toward its Cdk substrate resides within the catalytic domain itself and yet is in a region(s) that is outside the phosphate binding site of the enzyme.     

Diagnosis of infantile and juvenile forms of GM2 gangliosidosis variant 0. residual activities toward natural and different synthetic substrates

Summary p-Nitrophenyl-6-sulfo-2-acetamido-2-deoxy--d-glucopyranoside, which is known to be a specific substrate for human hexosaminidase A, has recently been used successfully for diagnosis of variants B and B¹ of G_M2-gangliosidosis (Fuchs et al. 1983; Kytzia et al. 1983; Li et al. 1983). However, it is hydrolyzed by hexosaminidase S as well and is therefore not suitable for detection of patients with variant 0, who reach the normal range of activity toward this substrats. Assay of ganglioside G_M2 cleaving activity in fibroblast extracts in the presence of the natural G_M2 activator protein reveals residual hexosaminidase A activities of less than 2% of normal controls in two infantile and up to 7.5% in two juvenile patients with variant 0.     

Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic beta-galactosidase substrates.

Current methods for detecting lacZ expression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced. To overcome this problem, we developed a series of new substrates for detection of lacZ expression in living cells under standard culture or physiological conditions. After incubation with these fluorogenic substrates, cultured lacZ-positive mammalian cells appear morphologically normal, continue to divide, and retain the fluorescent product. Because the product is so well retained, fluorescence intensity can be quantitatively related to the level of gene expression. We have demonstrated this correlation using transformed yeast cells bearing various plasmids, each containing the lacZ gene and a unique promoter sequence with known capabilities for promoting gene expression in yeast.     

Co-production of alpha-amylase and beta-galactosidase by Bacillus subtilis in complex organic substrates

Various nutrients belonging to three categories, carbon, organic nitrogen and complex organic sources, were investigated for the first time in terms of their effect on the co-production of extracellular thermostable alpha-amylase and beta-galactosidase by Bacillus subtilis, a bacterium isolated from fresh sheep's milk. Among the organic nitrogen sources tested, tryptone and corn steep liquor favored their production. Substitution of soluble starch by various starchy substrates, such as corn flour, had a positive effect on both enzyme yields. Furthermore, a two-fold higher production of both enzymes was achieved when corn steep liquor or tryptone was used in combination with the different flours. Among the divalent cations examined, calcium ions appeared to be vital for alpha-amylase production. The crude alpha-amylase and beta-galactosidase produced by this B. subtilis strain exhibited maximal activities at 135 degrees C and 65 degrees C, respectively, and were also found to be significantly stable at elevated temperatures.     

In vitro inhibition of human cytochrome P450-mediated metabolism of marker substrates by natural products.

Spices, herbal and black teas, and soybean products were analyzed for their capacity to inhibit in vitro metabolism of drug marker substrates by human cytochrome P-450 (CYP) isoforms. Inhibition of drug metabolism was determined using aliquots or infusions from these products in a fluorescence-detection assay. Aliquots and infusions of all natural product categories inhibited 3A4 metabolism to some extent. Of the 26 aliquots from teas and spices further tested with 2C9, 2C19 and 2D6, many demonstrated significant inhibitory activity on the metabolism mediated by these isoforms. Black teas and herbal tea mixtures were generally more inhibitory than single-entity herbal teas. Spices and single-entity herbal teas showed species-specific isoform inhibition with sage, thyme, cloves, St John's Wort and goldenseal having the highest activity against several isoforms. Seven soybean varieties tested, as well as daidzein and genistein isolated from soybean, were found to inhibit 3A4-mediated metabolism. Genistein was found to inhibit 3A7- but not 3A5-mediated metabolism of the marker substrate. Assessment of the in vitro CYP inhibition potential for these natural products has important implications for predicting the likelihood of natural product-drug interactions if these products are taken concomitantly.     

Structural characterization of lactotetraosylceramide, a novel glycosphingolipid isolated from human meconium.

In the course of work on a systematic structural mapping of nonacid glycosphingolipids of human meconia, special attention was given to a major component preliminarily identified as an isomer of neolactotetraosylceramide (paragloboside). This component was isolated in its pure form from meconium of a blood group O individual and subjected to detailed structural analyses, using mass spectrometry and proton NMR spectroscopy on intact permethylated and permethylated-reduced (LiAlH4) derivatives, and gas liquid chromatography on degradational products of native, permethylated, and permethylated-reduced derivatives. The isolated compound was conclusively shown to have the structure Galp beta 1 yields 3GlcNAcp beta 1 yields 3Galp beta 1 yields 4Glcp beta 1 yields 1Cer, and is thus identified as lactotetraosylceramide. The major fatty acids were 2-hydroxy fatty acids with 16 and 20 to 24 carbon atoms, and the bases were sphingosine and phytosphingosine. This glycolipid, although not isolated and structurally characterized before, has long been thought of as a precursor substance of the Lewis active glycolipids and of ABH-active glycolipids with a type 1 saccharide chain.     

The human blood fluke Schistosoma mansoni synthesizes a novel type of glycosphingolipid.

Previous studies have shown that the glycoprotein oligosaccharides synthesized by adult Schistosoma mansoni, the organism responsible for human schistosomiasis, are unusual in that they contain terminal beta-GalNAc residues and lack sialic acid. These observations and other studies indicating that schistosome glycoproteins and glycolipids are antigenic in infected animals led us to investigate the structures of the glycosphingolipids synthesized by these organisms and to determine whether they are structurally related to those synthesized by their vertebrate hosts. For our studies, adult schistosomes were metabolically radiolabeled with either [3H]galactose or [3H]glucosamine, and the newly synthesized glycosphingolipids were isolated and characterized. The major glycosphingolipids synthesized by adult schistosomes were found to be galactosylceramide and glucosylceramide. The adult worms synthesized no lactosylceramide (Gal beta 1-4Glc-ceramide), a common constituent of vertebrate cells; however, another disaccharide-containing glycosphingolipid cleavable by ceramide glycanase was found. The results of compositional and methylation analyses and exoglycosidase treatments demonstrated that this ceramide-disaccharide has the structure GalNAc beta 1-4Glc-ceramide. We also found that extracts of adult schistosomes are unable to transfer Gal from UDP-Gal to glucosylceramide, whereas extracts of Chinese hamster ovary cells, as a control, are able to do so, confirming that schistosomes are unable to synthesize lactosylceramide. Low levels of higher molecular weight glycosphingolipids were also found to be synthesized by adult schistosomes, and although their levels were too small to allow definitive characterization, compositional analyses indicated that they also contained GalNAc. We have tentatively designated the new disaccharide structure GalNAc beta 1, 4Glc- the "schistocore", which may represent a new type of glycosphingolipid core series.     

Regional localization of a beta-galactosidase locus on human chromosome 22.

Human white blood cells with an X/22 translocation [46, XX, t(X;22)(q23;q13)] were fused with Chinese hamster cells. The isolated hybrids were analyzed for human chromosomes and 21 enzyme markers. An electrophoretic technique for studying the beta-galactosidase isoenzymes in man-Chinese hamster hybrid cells was developed. Immunological studies showed that the beta-galactosidase marker studied in these hybrids did contain immunological determinants of human origin. Furthermore the results provided evidence that a locus for beta-galactosidase is situated on chromosome 22 distal to the breakpoint in q13.