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PubMed1996

Cloning, expression and characterization of a blood group B active recombinant alpha-D-galactosidase from soybean (Glycine max).

A cDNA encoding soybean alpha-D-galactosidase [E.C. 3.2.1.22] was obtained by screening a soybean library with Phaseolus alpha-D-galactosidase cDNA. The Glycine max alpha-D-galactosidase cDNA is 1.75 kb long and contains untranslated 5' and 3' sequences. The deduced amino acid sequence of the soybean gene has a high degree of homology with other eucaryotic alpha-D-galactosidases. Recombinant alpha-D-galactosidase (rGal) was expressed in Pichia pastoris and purified by affinity chromatography. Purified rGal was homogeneous as judged by SDS-PAGE analysis with the relative molecular mass under reducing conditions of 39.8, and under nonreducing conditions 38.0 kDa. The expressed protein contained the sequence NGLGHTPPMG at the N-terminus, corresponding to the deduced amino acid sequence of the soybean gene. The relative native molecular mass by Sephacryl S-200 chromatography was determined to be 33.1 kDa. The specific activity was 295.6 mumoles of PNP-alpha-D-galactopyranoside hydrolyzed per mg pure rGal per min. rGal was highly specific for alpha-D-galactosyl residues. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen in native human erythrocyte cell suspension assays. The only detectable erythrocyte phenotypic change was loss of the B and P1 epitopes. Consequently, recombinant Glycine max alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of universally transfusable type O erythrocytes by enzymatic conversion.

ABO Blood-Group System↗

PubMed2000

Impaired elastic-fiber assembly by fibroblasts from patients with either Morquio B disease or infantile GM1-gangliosidosis is linked to deficiency in the 67-kD spliced variant of beta-galactosidase.

We have previously shown that intracellular trafficking and extracellular assembly of tropoelastin into elastic fibers is facilitated by the 67-kD elastin-binding protein identical to an enzymatically inactive, alternatively spliced variant of beta-galactosidase (S-Gal). In the present study, we investigated elastic-fiber assembly in cultures of dermal fibroblasts from patients with either Morquio B disease or GM1-gangliosidosis who bore different mutations of the beta-galactosidase gene. We found that fibroblasts taken from patients with an adult form of GM1-gangliosidosis and from patients with an infantile form, carrying a missense mutations in the beta-galactosidase gene-mutations that caused deficiency in lysosomal beta-galactosidase but not in S-Gal-assembled normal elastic fibers. In contrast, fibroblasts from two cases of infantile GM1-gangliosidosis that bear nonsense mutations of the beta-galactosidase gene, as well as fibroblasts from four patients with Morquio B who had mutations causing deficiency in both forms of beta-galactosidase, did not assemble elastic fibers. We also demonstrated that S-Gal-deficient fibroblasts from patients with either GM1-gangliosidosis or Morquio B can acquire the S-Gal protein, produced by coculturing of Chinese hamster ovary cells permanently transected with S-Gal cDNA, resulting in improved deposition of elastic fibers. The present study provides a novel and natural model validating functional roles of S-Gal in elastogenesis and elucidates an association between impaired elastogenesis and the development of connective-tissue disorders in patients with Morquio B disease and in patients with an infantile form of GM1-gangliosidosis.

Alternative Splicing↗

PubMed2001

Molecular cloning of beta-galactosidase from Japanese pear (Pyrus pyrifolia) and its gene expression with fruit ripening.

We have cloned a cDNA fragment encoding a beta-galactosidase from Japanese pear (Pyrus pyrifolia) fruit (JP-GAL). It contained an untranslated sequence of 182 nucleotides at the 5' end, a presumptive coding sequence of 2,193 nucleotides and an untranslated sequence of 268 nucleotides including a polyadenylation signal and a poly (A) tail at the 3' end. It encoded a protein with a calculated molecular weight of 80.9 kDa which consists of 731 amino acids. Both the nucleotide and the deduced amino acid sequences showed a 98% sequence identity with that obtained from the apple beta-galactosidase cDNA. The peptide sequence obtained from the purified Japanese pear beta-galactosidase III matched the deduced amino acid sequence of SVSYDHKAIIINGQKRILISG (amino acid 25-45). Northern blot analysis showed that the probe derived from JP-GAL hybridized to a single 2.6 kb RNA. The mRNA was detected solely in the fruit; none was detected in the buds, leaves, roots or shoots of the Japanese pear. The steady-state level of the beta-galactosidase mRNA was measured during fruit ripening in three cultivars, Housui, Kousui (early ripening) and Niitaka (late ripening). The results showed that regardless of the cultivar, no JP-GAL mRNA was detected in the immature fruit. Increment of the mRNA level with fruit ripening coincided with the increase in the beta-galactosidase III activity. Our results showed that the expression of JP-GAL correlated with fruit softening and JP-GAL may be beta-galactosidase III.

Amino Acid Sequence↗

PubMed1992

A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-beta-galactosidase.

The lacG gene encoding the 6-phospho-beta-galactosidase (E.C.3.2.1.85) of Staphylococcus aureus was fused to the protein A gene in the plasmid pRIT2T. Escherichia coli cells containing this plasmid produce a fusion protein with both IgG binding and 6-phospho-beta-galactosidase activities after heat induction. The recombinant gene was overexpressed and the hybrid protein was purified to homogeneity in high yield. The chimeric protein was shown to have almost identical enzymatic characteristics to pure 6-phospho-beta-galactosidase. This result leads to the conclusion that a free N-terminus of the 6-phospho-beta-galactosidase is not required for biological activity. The hybrid protein of protein A and 6-phospho-beta-galactosidase was used as an enzyme conjugate in enzyme-linked immunosorbent assays (ELISA). The experiments presented demonstrate that the 6-phospho-beta-galactosidase is a suitable fusion partner in various diagnostic applications where an unique biological activity is required.

Amino Acid Sequence↗

PubMed1995

Two genes encoding the beta-galactosidase of Lactobacillus sake.

The beta-galactosidase of Lactobacillus sake DSM 20017 is encoded by two genes located on its chromosome. These genes designated lacL and lacM were cloned in Escherichia coli NM 554 on an 8.65 kbp HindIII fragment inserted in vector pRB473. Deletion analysis of the originally cloned fragment revealed that both genes are required for the formation of a functional beta-galactosidase. lacL and lacM are transcribed as a single transcript of approximately 2.9 kbp starting 34 bp upstream of the translational start codon. The proteins derived from lacL and lacM share only 18-59% homology with other beta-galactosidases. The genes encoding the beta-galactosidase are scattered with multiple direct and inverted repeats of 9-12 bp. However, comparison with the plasmid-encoded Leuconostoc lactis beta-galactosidase revealed equal distribution of conserved amino acid residues and suggests that the genes have a common origin. Specific deletions or insertions resulting from the presence of the repeats were not observed. The L. sake beta-galactosidase was phenotypically expressed in E. coli NM 554 and Lactobacillus curvatus LTH 1432. Its two genes can be used to replace antibiotic reporter genes to develop food-grade vectors and alpha-complementation systems for self-cloning in meat lactobacilli.

Amino Acid Sequence↗

PubMed1999

Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between beta-galactosidase and other glycohydrolases.

Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an alpha/beta barrel. Those that are most similar to the alpha/beta barrel of E. coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases. The structure comparison suggests that beta-amylase should also be included in this family. Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.

Animals↗

PubMed1994

Hydrolysis of lactosylceramide by human galactosylceramidase and GM1-beta-galactosidase in a detergent-free system and its stimulation by sphingolipid activator proteins, sap-B and sap-C. Activator proteins stimulate lactosylceramide hydrolysis.

Two exo-beta-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-beta-galactosidase (EC 3.2.1.23) and galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap-B and sap-C, rather than detergents, to stimulate the reaction. While sap-B was a better activator for the reaction catalyzed by GM1-beta-galactosidase, sap-C preferentially stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2 > C4 > C6 > C8 > C10 > C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-beta-galactosidase and sap-C-stimulated degradation of liposomal galactosylceramide by galactosylceramidase. Kinetic and dilution experiments indicated that sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-beta-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561-572]. GM1-beta-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap-C and sap-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.

Antigens, CD↗

PubMed2000

alpha-Galactosidase A from Pseudomonas fluorescens subsp. cellulosa: cloning, high level expression and its role in galactomannan hydrolysis.

A library of Pseudomonas fluorescens subsp. cellulosa genomic DNA, constructed in lambda ZAPII, was screened for alpha-D-galactosidase activity. The DNA inserts from six galactosidase-positive clones were rescued into plasmids. Restriction digestion and Southern analysis revealed that each of the plasmids contained a common DNA sequence. The sequence of the Pseudomonas DNA in one of the plasmids revealed a single open reading frame (aga27A) of 1215 bp encoding a protein of M(r) 45900, designated alpha-galactosidase 27A (Aga27A). Aga27A exhibited extensive sequence identity with alpha-galactosidases in glycoside hydrolase 27, and appeared to be a single domain protein. The recombinant alpha-galactosidase was expressed at high levels in Escherichia coli and the biophysical properties and substrate specificity of the enzyme were evaluated. The data showed that Aga27A was a mesophilic neutral acting non-specific alpha-galactosidase. Both P. fluorescens subsp. cellulosa mannanase A (ManA) and Aga27A hydrolyse the polymeric substrate, carob galactomannan. Sequential hydrolysis with AgaA followed by ManA, or ManA followed by AgaA enhanced product release. The positive effects of sequential hydrolysis are discussed.

Amino Acid Sequence↗

PubMed2005

Delivery of beta-galactosidase to mouse brain via the blood-brain barrier transferrin receptor.

Enzyme replacement therapy of lysosomal storage disorders is complicated by the lack of enzyme transport across the blood-brain barrier (BBB). The present studies evaluate the delivery of a model enzyme across the BBB following enzyme conjugation to a BBB receptor-specific monoclonal antibody (mAb). Bacterial beta-galactosidase (116 kDa) was conjugated to the rat 8D3 mAb to the rat transferrin receptor (TfR) via a streptavidin-biotin linkage. The unconjugated beta-galactosidase or the beta-galactosidase-8D3 conjugate was injected intravenously in adult mice, and enzyme activity was measured at 1 and 4 h in brain and peripheral organs (liver, spleen, kidney, and heart). Unconjugated beta-galactosidase was rapidly removed from the blood compartment owing to avid uptake by liver and spleen. There was minimal uptake of the unconjugated beta-galactosidase by brain. Following conjugation of the enzyme to the 8D3 TfRmAb, there was a 10-fold increase in brain uptake of the enzyme based on measurement of enzyme activity. Histochemistry of brain showed localization of the enzyme in the intraendothelial compartment of brain following intravenous injection of the enzyme-mAb conjugate. The capillary depletion technique showed that more than 90% of the enzyme-8D3 conjugate that entered into the endothelial compartment of brain passed through the BBB to enter brain parenchyma. In conclusion, high molecular weight enzymes, such as bacterial beta-galactosidase, can be conjugated to BBB targeting antibodies for effective delivery across the BBB in vivo. Fusion proteins comprised of BBB targeting antibodies and recombinant enzymes could be therapeutic in the treatment of the brain in human lysosomal storage disorders.

Animals↗

PubMed1994

Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase.

Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.

Amino Acid Sequence↗

PubMed1992

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes.

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.

Amino Acid Sequence↗

PubMed1995

Efficient selection of recombinant adenoviruses by vectors that express beta-galactosidase.

Adenovirus serotype 5 vectors which contain the Excherichia coli beta-galactosidase gene driven by the cytomegalovirus immediate-early promoter as a screenable marker have been made and successfully used in the construction of recombinant adenoviruses. The beta-galactosidase gene has been introduced into viruses in which the E3 region is maintained or deleted and in which the cis-acting packaging sequence has been reiterated at the right end of the chromosome. A unique BstBI site has been introduced 3' of the beta-galactosidase gene. Cotransfection of BstBI-digested vector DNA and a plasmid containing the left end of the viral chromosome followed by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) results in clear plaques when overlap recombination has occurred and blue plaques when ligation of the viral arms has occurred within the host cell. The beta-galactosidase-expressing viruses grow to lower titers than do the parental viruses, leading to a relative growth advantage for viruses resulting from overlap recombination. Combined with color selection based on the beta-galactosidase gene, this system permits efficient production and selection of recombinant viruses after cotransfection of BstBI-digested viral DNA with a plasmid including left-end viral sequences and the gene of interest. The beta-galactosidase-expressing viral DNAs were used to construct viruses containing BstBI sites on either side of the cis-acting packaging element as a means of testing their utility.

Adenoviridae↗

PubMed1993

Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies.

A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.

Aeromonas↗

PubMedDate not supplied

The properties of beta-galactosidases (Escherichia coli) with halogenated tyrosines.

An Escherichia coli tyrosine auxotroph (MR1) with an inducible lacZ was generated by mutagenesis. Of several tyrosine derivatives tested, only m-fluorotyrosine supported the growth of this mutant and allowed synthesis of active beta-galactosidase. The pH profiles of the beta-galactosidase that was obtained when this mutant was grown on m-fluorotyrosine (81.5% of the tyrosine was replaced by m-fluorotyrosine) indicated that a tyrosine may be acting as a general acid-base catalyst and that it (or another tyrosine with the same pKa) may be involved in substrate binding. Inactivation of normal beta-galactosidase by treatment with lactoperoxidase in the presence of I- did not affect affinity-column binding, but incubation of this iodinated beta-galactosidase with chymotrypsin caused a rapid degradation of a portion of the treated enzyme equal to the portion of the activity that was lost. A study with 125I- showed that the rapid degradation was mainly confined to iodinated molecules of enzyme. These studies indicate that iodination of beta-galactosidase does not affect binding ability, but causes the enzyme to lose catalytic activity and become susceptible to chymotryptic action. Chloroperoxidase also caused rapid inactivation of normal beta-galactosidase in the presence of Br- or I-, but there was a lag followed by a slow inactivation in the presence of Cl-.

Binding Sites↗

PubMed1975

Hormonal regulation of isoenzymes of N-acetyl-beta-glucosaminidase and beta-galactosidase during spermatogenesis in the rat.

Isoenzymes of beta-galactosidase and of N-acetyl-beta-glucosaminidase were assayed during development of rat testis and as a function of hormonal treatments. Isoenzyme 1 of beta-galactosidase was highest in specific activity in the 4-day-old testis, at a point when Sertoli cells and gonocytes were the predominant cell type. Beta-galactosidase II, previously shown to be associated with the sperm acrosome, was undetectable through the spermatocyte stage of development, but increased in specific activity during the formation of spermatids. The specific activities of isoenzymes I and II of N-acetyl-beta-glucosaminidase increased markedly in association with the formation of spermatogonia and spermatocytes, and then declined with the appearance of spermatids. Following hypophysectomy of rats at 26 days of age or in adulthood the specific activities of the lysosomal enzymes beta-galactosidase I and N-acetyl-beta-glucosaminidase I and II increased markedly, while the acrosomal beta-galactosidase II was undetectable. The normal patterns of isoenzyme distributed were restored completely by administration of LH and FSH or testosterone to hypophysectomized animals. These results thus demonstrate specific patterns of isoenzyme concentration during spermatogenesis. Formation of the acrosome in developing spermatids is associated with the induction of new forms of beta-galactosidase (isoenzyme II) and N-acetyl-beta-glucosaminidase (sperm isoenzyme). These molecules appear to be specialized forms which may participate in fertilization, and their induction is dependent upon the actions of gonadotropins or testosterone.

Animals↗

PubMed2005

Reverse reaction of Aspergillus niger APC-9319 alpha-galactosidase in a supersaturated substrate solution: production of alpha-linked galactooligosaccharide (alpha-GOS).

The alpha-galactosidase that effectively catalyzes a reverse reaction of galactose, Aspergillus niger APC-9319 alpha-galactosidase, was screened from industrial enzyme preparations for food processing containing alpha-galactosidase activity. Reverse reaction of A. niger APC-9319 alpha-galactosidase was performed using a supersaturated solution (90% galactose [w/v]). A. niger APC-9319 alpha-galactosidase was not inhibited even in high substrate concentration, and effectively catalyzed the reverse reaction. The yield of the reaction product, alpha-linked galactooligosaccharide (alpha-GOS), increased greatly as the initial concentration of galactose increased to 90% (w/v), and was more than 50%. Furthermore, the half life of enzyme activity was about three times as long as that using 60% galactose (w/v). alpha-GOS (1.4 g) was prepared from galactose (3.0 g) by reverse reaction of A. niger APC-9319 alpha-galactosidase. The alpha-GOS contained 58% alpha-galactobiose (alpha-Gal2), 28% alpha-galactotriose, and 14% oligosaccharides larger than alpha-galactotriose. The main component of positional isomers in alpha-Gal2 was alpha-1,6Gal2.

Aspergillus niger↗

PubMed2004

Immunoquantification of alpha-galactosidase: evaluation for the diagnosis of Fabry disease.

BACKGROUND: Fabry disease is an X-linked inborn error of glycosphingolipid catabolism resulting from a deficiency of the lysosomal exoglycohydrolase, alpha-galactosidase. Enzyme replacement therapy is currently available for Fabry disease, but early diagnosis before the onset of irreversible pathology will be mandatory for successful treatment. Presymptomatic detection would be possible through the use of a newborn-screening program. We report on the use of sensitive assays for the measurement of alpha-galactosidase protein and activity and for the protein saposin C, which are diagnostic markers for Fabry disease. METHODS: Two sensitive immunoassays for the measurement of alpha-galactosidase activity and protein were used to determine the concentrations of alpha-galactosidase in dried filter-paper blood spots and plasma samples from control patients and patients with a lysosomal storage disorder (LSD). RESULTS: Fabry hemizygous individuals were clearly identified from control populations by decreases in both alpha-galactosidase activity and protein. Fabry heterozygotes generally fell between the hemizygotes and controls. Including the measurement of saposin C enabled differentiation between Fabry heterozygotes and controls. In blood spots, all Fabry individuals could be distinguished from control blood spots as well as from 16 other LSD patients. CONCLUSIONS: The determination of alpha-galactosidase activity or protein in dried filter-paper blood spots could be used for the diagnosis of Fabry patients. With further validation, these assays could be used for the identification of Fabry patients in newborn-screening programs and may also be suitable for screening high-risk populations.

Adult↗

PubMed2000

Viability and beta-galactosidase activity of dairy propionibacteria subjected to digestion by artificial gastric and intestinal fluids.

An important criterion to consider in the selection of strains for dietary adjuncts is the ability of the microorganisms to survive the severe conditions of acidity and bile concentrations usually found in the gastrointestinal tract. In the present work, we report the effects of digestions by artificial gastric and intestinal fluids on beta-galactosidase activity and survival of four strains of dairy propionibacteria previously selected by their bile tolerance and beta-galactosidase activity. The strains were exposed to artificial gastric juice at pH values between 2 and 7 and then subjected to artificial intestinal digestion. Both viability and beta-galactosidase activity were seriously affected at pH 2. Skim milk and Emmental cheese juice exerted a protective effect on the parameters tested. The trypsin present in the intestinal fluid inactivated the enzyme beta-galactosidase in strains of Propionibacterium freudenreichii but not in Propionibacterium acidipropionici. Moreover, the presence of bile salts enhanced the beta-galactosidase activity of these strains by permeabilization of the cells during the first hour of exposure. The intestinal transit rate confirmed the permanence of the bacteria in the intestine for long enough to be permeabilized. These results suggest that P. acidipropionici would be a good source of beta-galactosidase activity in the intestine. We also propose a practical and effective in vitro method as a tool of screening and selection of potential probiotic bacteria.

Animals↗