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PubMed2000

Structurally conserved soluble acid phosphatases are synthesized and released by Leishmania major promastigotes.

Previously it was reported that promastigotes of virtually all pathogenic Leishmania species, except Leishmania major, release a structurally conserved soluble acid phosphatase (AcP) activity during their growth in vitro (P. S. Doyle and D. M. Dwyer, Exp. Parasitol. 77, 435-444 1993). In the current study we used a highly sensitive fluorogenic detection method to demonstrate that soluble AcPs were in fact produced by promastigotes of several different strains of L. major. These L. major AcP activities were readily immunoprecipitated with a rabbit antibody previously generated against the L. donovani AcP. Results of metabolic labeling and immunoprecipitations demonstrated that AcPs produced by the L. majors strains examined had an apparent molecular mass of approximately 77 kDa. Results of Southern hybridization analysis with an L. donovani AcP gene probe showed that the AcP gene loci were conserved in the L. major strains examined. Taken together, these results indicate that the AcP enzyme has been structurally and functionally conserved throughout the evolution of pathogenic species of Leishmania. Such conservation suggests that the AcPs play a functional role in the growth and survival of this group of important human pathogens.

Acid Phosphatase↗
PubMed2001

Leishmania donovani: expression and characterization of Escherichia coli-expressed recombinant chitinase LdCHT1.

Leishmania parasites produce chitinase activity (EC. 3.2.1.14) thought to be important in parasite-sandfly interactions and transmission of the parasite to the vertebrate host. Previous observations have suggested that parasite chitinases are involved in degradation of the sandfly peritrophic matrix and the chitinous layer of the cardiac valve cuticle. This chitinase activity is thought to produce an incompetent pharyngeal valve sphincter and a route of egress that allow Leishmania promastigotes to be regurgitated into the site of blood feeding. In the studies reported here, enzymatically active L. donovani chitinase LdCHT1 was expressed as a thioredoxin fusion protein in Escherichia coli strain AD494 (DE3). Recombinant LdCHT1 had a predominantly endochitinase activity, in contrast to previous reports of both exo- and endochitinase activities in axenic culture supernatants of diverse Leishmania spp. promastigotes. The predominant endochitinase activity of recombinant LdCHT1 is consistent with the presumed function of the enzyme in disrupting chitinous structures in the sandfly digestive system to allow transmission.

Acetylglucosamine↗
PubMed1997

Fast and slow tracks in lysozyme folding: insight into the role of domains in the folding process.

The folding of lysozyme involves parallel events in which hydrogen exchange kinetics indicate the development of persistent structure at very different rates. We have monitored directly the kinetics of formation of the native molecule by the binding of a fluorescently labelled inhibitor, MeU-diNAG (4-methylumbelliferyl-N,N'-diacetyl-beta-D-chitobioside). The data show that native character monitored in this way also develops with different timescales. Although the rate determining step on the slow pathway (approximately 75% of molecules at pH 5.5, 20 degrees C) can be attributed to the need to reorganise structure formed early in the folding process, the data indicate that the rate determining step on the fast track (involving approximately 25% of molecules) involves the docking of the two constituent domains of the protein. In the fast folding track the data are consistent with a model in which each domain forms persistent structure prior to their docking in a locally cooperative manner on a timescale comparable to the folding of small single domain proteins.

Animals↗
PubMed1997

The crystal structure of the complex of concanavalin A with 4'-methylumbelliferyl-alpha-D-glucopyranoside.

Concanavalin A (Con A) is the best known plant lectin, with important biological properties arising from its specific saccharide-binding ability. Its exact biological role still remains unknown. The complex of Con A with 4'-methylumbelliferyl-alpha-D-glucopyranoside (alpha-MUG) has been crystallized in space group P2(1) with cell dimensions a = 81.62 A, b = 128.71 A, c = 82.23 A, and beta = 118.47 degrees. X-ray diffraction intensities to 2.78 A have been collected. The structure of the complex was solved by molecular replacement and refined by simulated annealing methods to a crystallographic R-factor value of 0.182 and a free-R-factor value of 0.216. The asymmetric unit contains four subunits arranged as a tetramer, with approximate 222 symmetry. A saccharide molecule is bound in the sugar-binding site at the surface of each subunit, with the nonsugar (aglycon) part adopting a different orientation in each subunit. The aglycon orientation, although probably determined by packing of tetramers in the crystal lattice, helps to characterize the orientation of the saccharide in the sugar-binding pocket. The structure is the best determined alpha-D-glucoside:Con A complex to date and the hydrogen bonding network in the saccharide-binding site can be described with some confidence and compared with that of the alpha-D-mannosides.

Binding Sites↗
PubMed1993

Non-radioactive detection of Mycobacterium tuberculosis LCR products in a microtitre plate format.

As part of the development of the ligase chain reaction (LCR) into a tool which can be used by a wide variety of researchers, we have investigated several analytical detection systems for the products of this amplification reaction. While early work with this technology has used gel electrophoresis to separate the LCR probes from the ligated product, solid phase capture techniques are also applicable, particularly when one of the probes is modified with a 'hook' such as biotin, and the adjoining probe modified with a detectable label. In this study we report a comparison of eight different non-radioactive detection techniques and discuss the analytical sensitivity of each. Detection with laser scanning fluorescent gel electrophoresis remains the most sensitive, with the assay described herein capable of detecting 100 molecules of the Mycobacterium tuberculosis insertion element IS6110 in a background of 4 micrograms of unrelated DNA. This method was followed closely by solid-phase capture and chemiluminescence detection which gave a sensitivity of 1000 molecules of IS6110. Fluorescence detection was approximately 10-fold less sensitive than chemiluminescence detection, and absorbance detection was a further 10-fold less sensitive than fluorescence detection. However, absorbance detection even at this level can still be useful for systems where visual interpretation is desired.

Alkaline Phosphatase↗
PubMed2001

Molecular cloning and expression of an alpha-mannosidase gene in Mycobacterium tuberculosis.

Mannose is a major component of glycolipids and glycoproteins of the cell envelope of M. tuberculosis (Mtb). However, the enzymes involved in the biosynthesis and catabolism of mannosylated glycans are largely unknown. We demonstrate alpha-mannosidase activity towards the fluorescent substrate 4-methylumberlliferyl-alpha-D-mannopyranoside (4MU-Man) in cell lysates of attenuated and virulent Mtb bacilli, with two-fold higher activity in the virulent strain Erdman. Mannosidase activity was optimal at pH 6.5, was not inhibited by deoxymannojirimycin (dMNJ), was mildly inhibited by swainsonine (SW) and stimulated two-fold by EDTA. GenBank BLAST analysis for sequences homologous to eukaryotic alpha-mannosidases revealed a 3.6 kb putative gene (Rv0648) in Mtb cosmid SCY20H10 (Acc# z92772), with strong homology (48%) to the rat ER/cytosolic alpha-mannosidase and containing signature sequences of class 2 mannosidases. By RT-PCR, gene Rv0648 was found differentially expressed, with lower expression during growth in A549 pneumocyte cultures. Gene Rv0648 was cloned, expressed in E. coli, and alpha-mannosidase activity in cell lysates determined. Expression of alphaMan-pET in E. coli cells resulted in an eight-fold increase in mannosidase activity toward 4-MU-Man, upon IPTG induction. Partial purification of the histidine-tagged Mtb mannosidase by metal chelation affinity chromatography, and analysis by SDS-PAGE, showed a protein with the predicted m.w. of 137.5 kDa. Enzyme assays of the column fractions showed alpha-mannosidase activity toward synthetic aryl-mannose substrates, in fractions enriched in the recombinant Mtb mannosidase. These results demonstrate that gene Rv0648 encodes an active alpha-mannosidase in Mtb.

Amino Acid Sequence↗
PubMed1996

Expression and purification of a recombinant "small" sialidase from Clostridium perfringens A99.

A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.

Aminopeptidases↗
PubMed1998

Substrate, inhibitor, or antibody stabilizes the Glu 119 Gly mutant influenza virus neuraminidase.

We have previously reported the isolation and characterization of an influenza virus variant with decreased sensitivity to the neuraminidase-specific inhibitor zanamivir. This variant, which has a mutation in the active site, Glu 119 Gly (E119G), has the same specific activity as the wild-type neuraminidase (NA), but is inherently unstable, as measured by loss of both enzyme activity and NC10 monoclonal antibody reactivity. However, despite the instability of the NA, replication of the virus in liquid culture is not adversely affected. We demonstrate here that in addition to enhanced temperature sensitivity the mutant NA was significantly more sensitive to formaldehyde and to specimen preparation for electron microscopy. Substrate, inhibitor, or monoclonal antibodies stabilized the NA against all methods of denaturation. These results suggest that the instability of the variant is primarily at the level of polypeptide chain folding rather than at the level of association of monomers into tetramers. Furthermore the presence of high levels of substrate, either cell or virus associated, may be sufficient to stabilize the NA during virus replication.

Animals↗
PubMed2000

Characterization of a membrane-associated protein implicated in visna virus binding and infection.

The identity of the cellular receptor(s) for visna virus, an ovine lentivirus, is currently unknown; however, previous studies from our laboratory have identified membrane-associated proteins expressed selectively in susceptible cells which bind visna virus. Moreover, a polyclonal antibody (2-23), raised against a 45-kDa visna virus binding protein, bound specifically to the surface of susceptible cells in immunofluorescence assays and significantly reduced binding of visna virus to cells (S. E. Crane et al., 1991, J. Virol., 65, 6137-6143). In this report we extend our studies of this antibody (2-23), showing both that 2-23 significantly reduces visna virus infection of susceptible cells and that 2-23 immunoprecipitates a putative protein complex consisting of a prominent 30-kDa protein, as well as the 45-kDa immunogen, specifically from radiolabeled virus-susceptible sheep cells. Further, we demonstrate that the 30-kDa protein is a membrane-associated proteoglycan substituted with a chondroitin sulfate glycosaminoglycan (GAG) chain(s) and that treatment of susceptible cells with an inhibitor of GAG synthesis significantly reduces visna virus production. Collectively, these data support a role for a proteoglycan in visna virus cell binding and infection.

Animals↗
PubMed1976

Arylsulfatases A and B in metachromatic leukodystrophy and Maroteaux-Lamy syndrome: studies with 4-methylumelliferyl sulfate.

Metachromatic leukodystrophy and Maroteaux-Lamy syndrome can be diagnosed by assay of leukocyte or fibroblast arylsulfatase A and B activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. The arylsulfatases are extracted into a 27000 x g supernatant by sonication in 0.9% sodium chloride and then separated with CM-32 on columns or in test tubes. In 0.05 M sodium acetate pH 6.0, arylsulfatase A is not absorbed while arylsulfatase B is retained by the resin. The arylsulfatase B is then eluted from the resin with 0.3 M sodium chloride. The arylsulfatase A activity obtained from normal leukocytes and fibroblasts is linear for the initial 10 minutes of the reaction, is stimulated 3-fold by 6 mM lead acetate and inhibited 80% by 0.24 mM silver nitrate. After separation with CM-32, the arylsulfatase B activity is stimulated 3-fold by Triton X-100 (0.1%). Arylsulfatase A but not arylsulfatase B is destroyed by heat (60 degrees). Both leukocyte and fibroblast arylsulfatase A activity was reduced to 11% of control values in metachromatic leukodystrophy. Essentially no arylsulfatase B activity was detected in cells from patients with Maroteaux-Lamy syndrome. Metachromatic leukodystrophy heterozygotes but not Maroteaux-Lamy syndrome heterozygotes can also be distinguished by this method. A heat inactivation technique utilizing the differential thermal stabilities of the two enzymes for diagnosis of patients with Marotezux-Lamy syndrome is also described. The advantages of these 4-methylumbelliferyl sulfate assay procedures over the p-nitrocatechol sulfate method of assay are greater sensitivity, selectivity for the desired enzyme and potential for use in large scale testing.

Arylsulfatases↗
PubMed1987

Enzyme-linked immunosorbent assay (ELISA) for metallothionein.

A heterogeneous, double-antibody, fluorometric enzyme-linked immunosorbent assay (ELISA) is described for the detection and quantitation of metallothionein(MT) The protocol uses the same immunological reagents as the radioimmunoassay(RIA) developed previously in this laboratory; fluorescence replaces radioactivity for detection of the reference antigen in the specific binding reaction. Present results indicate that the developed ELISA has approximately the same range of capability in detecting and quantitating MT as is characteristic of the RIA. The ELISA has the advantage that the time required to perform a typical assay is significantly less than that required for the RIA.

Animals↗
PubMed1994

Molecular genetic analysis of the nagH gene encoding a hyaluronidase of Clostridium perfringens.

A recombinant lambda phage was identified in a Clostridium perfringens genomic library by means of its ability to hydrolyse the fluorescent substrate 4-methyl-umbelliferyl-beta-D-glucosaminide, isolated and shown to encode an endo-beta-N-acetylglucosaminidase. This enzyme, NagH, is also known as hyaluronidase, or Mu toxin, a putative virulence factor which is likely to act on connective tissue during gas gangrene. Nucleotide sequence analysis allowed the primary structure to be deduced and showed hyaluronidase to be a large exported protein of 114,392 Daltons and an enzyme of this size, endowed with the corresponding activities, was partially purified from C. perfringens. Hyaluronidase seems to be organised into two domains, an N-terminal region comprising 700 amino acids bearing the active site and a 300-residue C-terminal segment, containing three copies of an extended motif. Two other reading frames, linked to nagH, also appear to encode proteins with sugar-binding motifs.

Acetylglucosamine↗
PubMed1984

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

p-Nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-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 GM2-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 substrate. Assay of ganglioside GM2 cleaving activity in fibroblast extracts in the presence of the natural GM2 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.

Acetylglucosamine↗
PubMed1987

Hydrolysis of 4-methylumbelliferyl sulfate in periportal and pericentral areas of the liver lobule.

4-Methylumbelliferyl sulfate was used to characterize sulfatase activity in periportal and pericentral regions of the liver lobule in the perfused rat liver. Following infusion of 1.5 mM of this organic sulfatester, free 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were formed at rates of 13 and 9 mumoles/g/h, respectively, in livers from fasted, phenobarbital-treated rats. 5-Pregnen-3 beta-ol, 20-one sulfate inhibited hydrolysis and metabolite production completely, whereas perfusion with nitrogen-saturated perfusate or FCCP decreased total metabolite formation by only 30%. 4-Methylumbelliferone formed from the hydrolysis of 4-methylumbelliferyl sulfate was monitored with micro-light guides placed on periportal and pericentral areas of the liver lobule. Detection of the desulfated product was always greater in the downstream region, i.e., infusion of 4-methylumbelliferyl sulfate produced a higher fluorescence signal in pericentral areas when perfusion was in the anterograde direction, while periportal areas demonstrated higher activity during perfusion in the retrograde direction. Perfusion with nitrogen-saturated perfusate abolished these differences. Taken together, these data suggest that uptake of organic sulfateesters is partially energy dependent, follows the hepatic oxygen gradient inversely, and is a major rate determinant for sulfatase activity in the liver.

Animals↗
PubMed1996

Identification and partial characterization of three chitinase forms in Entamoeba invadens with emphasis on their inhibition by allosamidin.

Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a soluble fraction by anionic exchange, size exclusion and hydroxyapatite adsorption chromatographies. The enzymes, named here as A, B and B', showed molecular weights of 64, 33.4 and 33.4 kDa, respectively, as measured by gel filtration. Comparison of their levels of specific activity in partially purified samples revealed chitinase A as the major species. Chitinase B' was a minor component of the chitinolytic complex. Whereas some properties were common to the three forms, analysis of other parameters revealed significant catalytic site-related differences. Accordingly, the three chitinases hydrolyzed the fluorogenic substrate 4-methylumbelliferyl chitotriose with typical Michaelian kinetics and Km values of 4.5, 11.8 and 3.8 microM for A, B and B', respectively. Allosamidin strongly inhibited the three enzyme forms with different kinetics. Dixon plots revealed competitive-type inhibition and Ki values of 10.0, 2.3 and 10.8 nM for A, B and B', respectively. Km/Ki ratios indicated 450-, 350- and 5130-fold higher affinity for the inhibitor over the substrate for the A, B and B' forms, respectively. Results are discussed in terms of the possibility that the three chitinase species correspond to different enzyme proteins.

Acetylglucosamine↗
PubMed1993

A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C).

Both the alpha- and beta-anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA:glucosaminide N-acetyltransferase. Using the beta-anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed < 1% of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-glucosaminide requires the sequential action of the N-acetyltransferase and beta-hexosaminidase. Normal beta-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell extracts with a beta-hexosaminidase deficiency, however, a second incubation in the presence of excess beta-hexosaminidase is needed to avoid underestimation of the N-acetyltransferase activity.

Acetyltransferases↗