Determining the evolutionary potential of a gene

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base-substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta-galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.     

Effect of bile salts on lactosylceramide beta-galactosidase activities in human brain, liver and cultured skin fibroblasts

The effect of bile salts on the hydrolysis of lactosylcermide by human beta-galactosidases in vitro was studied using cultured skin fibroblasts, liver and brain tissue. The evidence for two distinct enzymes that can catalyze the hydrolysis of lactosylceramide was observed when the bile salt was changed from pure sodium taurocholate to either crude taurocholate, or pure glycodeoxycholate, taurodeoxycholate or taurochenodeoxycholate. Tissues from patients with Krabbe's disease were found to be deficient in lactosylceramide beta-galactosidase activity (lactosylceramidase I) when pure taurocholate was used in the assay. When crude taurocholate was used in the assay, the Krabbe patients appeared to have normal activity for this enzyme. In place of crude taurocholate the pure salts of glycodeoxycholate, taurodeoxycholate and taurochenodeoxycholate worked even better to stimulate the second lactosylceramide beta-galactosidase activity and GM1 gangliosidosis patients exhibiting little if any activity. Therefore, lactosylcermidase I is stimulated by crude taurocholate or pure glycodeoxycholate, taurodeoxycholate and taurochenodeoxycholate. The use of pure bile salts to assay lactosylceramidase I and II will result in better reproducibility for these enzyme activities between laboratories.     

Characterization of a strictly specific acid beta-galactosidase from Achatina achatina

An acid beta-galactosidase was isolated from the digestive juice of Achatina achatina and purified to homogeneity by anion exchange, gel-filtration and hydroxyapatite chromatographies. This enzyme is soluble, as are the cytosolic beta-galactosidases, functions at acid pH like the lysosomal enzymes but differs from the other soluble animal beta-galactosidases in that it is highly specific for the beta-D-galactosyl residue. In addition, it cleaves the beta1-4 linkage much faster than the beta1-3 and beta1-6 linkages. The enzyme is a monomeric glycoprotein with a molecular mass of 120-125 kDa and the carbohydrate moiety makes up approximately 6% (w/w) of the protein. The amino acid composition displays an important amount of acidic/amide and hydroxy amino acid residues and a low content of basic residues. The enzyme activity is markedly affected by the ionic strength of the medium and the rate-pH curve was shifted towards higher pH values in the presence of added salt. Acid beta-galactosidase is capable of catalysing transgalactosylation reactions. The yields of galactosylation of hydroxy amino acid derivatives, catalysed by the enzyme in the presence of lactose as the glycosyl donor, were higher than those reported previously with conventional sources of beta-galactosidases. In addition, the pH optimum is different for the hydrolysis (pH 3.2) and transgalactosylation (pH 5.0) reactions. On the basis of this work, the enzyme could be used as a tool in the structural analysis of D-galactose-containing oligosaccharide chains, as well as for the synthesis of glycoconjugates.     

Usefulness of plasma catecholamines during head-up tilt as a measure of sympathetic activation in vasovagal patients

We purified and characterized a thermophilic beta-galactosidase from Thermus sp. A4 isolated from the Atagawa hot spring (Shizuoka, Japan). The enzyme was monomeric, and its molecular mass was estimated to be 75 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was extremely thermostable and retained its full activity after incubation at 70 degrees C for 20 h. The Km observed were 5.9 mM for ortho-nitrophenyl beta-D-galactopyranoside and 19 mM for lactose. We cloned and analyzed the complete sequence of the gene encoding this enzyme. It was found to consist of 645 amino acid residues. We propose that this enzyme and seven other unclassified beta-galactosidases are new members of family 42 of the glycosyl hydrolases.     

Regioselectivity of the enzymatic transgalactosidation of D- and L-xylose catalysed by beta-galactosidases

The regioselectivity of enzymatic transgalactosidation depends on the source of the beta-galactosidase used. When the galactosyl acceptor only contains secondary hydroxyl groups, e.g., D- or L-xylose, it is possible to find an enzyme that catalyses preferentially the synthesis of any of the three regioisomers 4-, 3- and 2-O-beta-D-galactopyranosyl-D-xylose (1, 2 and 3, respectively) or 4-, 3- and 2-O-beta-D-galactopyranosyl-L-xylose (4, 5 and 6, respectively). Enriched mixtures in 1, 2 or 3 were obtained using beta-galactosidases from Escherichia coli, bovine testes or Aspergillus oryzae, respectively, by transgalactosidation reaction of O-nitrophenyl-beta-D-galactopyranoside and D-xylose, and enriched mixtures in 4, 5 or 6 were obtained in a similar way using beta-galactosidases from Aspergillus oryzae, lamb small-intestine (intestinal lactase-phloridzin hydrolase) or Saccharomyces fragilis, respectively, using L-xylose as acceptor.     

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.     

Interaction of estradiol, progesterone and corticosterone on uterine connective tissue degrading enzymes

The impact of ovarian hormones and corticosterone acetate on uterine connective tissue degrading enzymes were studied in mature albino rats. Ovariectomy resulted in a significant increase in the activities of alpha- and beta-galactosidases and glucosidases in the uterus. Administration of estradiol to ovariectomized rats brought back the activities of alpha-galactosidase and alpha-glucosidase to normalcy. While beta-galactosidase and beta-glucosidase were significantly decreased. Administration of progesterone to ovariectomized rats resulted in the increase of alpha- and beta-galactosidases and glucosidases. Administration of corticosterone to ovariectomized rats produced a further increase in alpha- and beta-galactosidases and glucosidases in the uterus. Adrenalectomy in ovary intact rats produced a decrease in alpha-galactosidase however, beta-glucosidase was significantly increased. Administration of corticosterone to ovary intact rats significantly increased the activities of alpha- and beta-galactosidases, while alpha- and beta-glucosidases were found to be decreased. Ovariectomy resulted in a significant increase in the activities of cathepsin-D and cathepsin-E. Administration of estradiol to ovariectomized rats brought back the activity of cathepsin-D to normalcy, whereas cathepsin-E was significantly increased. Administration of progesterone as well as estradiol to ovariectomized rats significantly increased the levels of cathepsin-E, however, cathepsin-D was brought back to normalcy. Administration of corticosterone to ovariectomized rats as well as ovariectomy + adrenalectomy significantly increased the activity of cathepsin-D and cathepsin-E. Adrenalectomy significantly decreased the activity of cathepsin-D, while administration of corticosterone increased the cathepsin-D and cathepsin-E in the uterus. Therefore, these results suggest that estradiol is a potent ovarian steroid protecting the extra cellular matrix components. The effect of progesterone appears to modulate and act hand in hand with estradiol. Corticosterone appears to have an opposite effect to that of estradiol.     

Immunochemical characterization of hepatic cytochrome P450 isozymes in the channel catfish: assessment of sexual, developmental and treatment-related effects

The profiles of immunoreactive proteins recognized by antibodies raised against purified trout P-450 isoforms (CYP1A1, CYP2M1 and CYP2K1) were examined in channel catfish liver by Western blot analysis. Gender differences in basal expression of these isoforms, as well as responses to known inducers of mammalian isoforms (ethanol, beta-naphthoflavone and clofibric acid) and early life stage (3 and 6 months) profiles are described. Two similar protein bands were detected by Western blotting in mature untreated catfish with CYP2K1 and CYP2M1 antibodies. A third band is detected by anti-2K1 in fish treated with beta-naphthoflavone; this band was verified as CYP1A, with about twice the level of expression in males versus females. No difference between sexes was seen in the expression of the 51-kDa CYP2-reactive bands; however, a significant difference (female > male) was seen in the lower molecular weight CYP2 band (47-kDa). Ethanol treatment caused a dose-dependent decrease in the 47-kDa CYP2-reactive isoforms but no change in the 51-kDa band. Clofibric acid treatment caused an increase in both the 51-kDa CYP2 protein as well as in liver somatic index. Age-dependent changes in isoform expression were also detected in CYP2-reactive forms, with a novel protein (53-kDa) detected in 3-month-old fish. The results from this study provide insight into the regulation of constitutive catfish CYP isoforms and prepares a foundation for further examination of the biotransformation capabilities of an important aquatic species.     

Improved mimicry of a foot-and-mouth disease virus antigenic site by a viral peptide displayed on beta-galactosidase surface

A major antigenic site (site A) of foot-and-mouth disease virus includes multiple overlapping epitopes located within the flexible G-H loop of capsid protein VP1. We have studied the antigenicity of several recombinant E. coli beta-galactosidases displaying the site A from a serotype C virus in different surface regions of the bacterial enzyme. In each one of the explored insertion sites, the recombinant peptide shows different specificity with a set of anti-virus monoclonal antibodies directed to site A. In some of them, the inserted stretch mimics better than free or haemocyanin-coupled peptide the antigenicity of site A in the intact virus. In particular, an insertion within an exposed loop involved in the activating interface of beta-galactosidase (amino acids 272 to 287) led to a significant improvement of the overall reactivity. Since insertions at this site renders proteins enzymatically active, the activating interface could be an adequate place for the presentation of foreign antigens in correctly assembled beta-galactosidase tetramers. These results also suggest that anti-virus antibodies directed against the major antigenic site of FMDV recognize different conformations of the G-H loop, which are better reproduced in some of the recombinant proteins because of the dissimilar restrictions imposed by each particular insertion site.     

Successful reconstitution of human hematopoiesis in the SCID-hu mouse by genetically modified, highly enriched progenitors isolated from fetal liver

Highly purified CD34++CD38-Lin- hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli beta-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the beta-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38-Lin- cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.     

Study on structural gene expression in human insulinoma

A human insulinoma cDNA library was constructed in the expression plasmid vector pUEX1. The clone pUEX1Ins12 was selected by means of hybridization with an insulin probe. It codes for full size amino acid sequence preproinsulin. The bacterial strain pUEX3Ins8 producing proinsulin as beta-galactosidase fusion protein was obtained for the use of recombinant protein as an antigen in an ELISA to detect serum antibodies in subjects with IDDM. Recombinant clones containing the middle, N- and C-terminal domains of the GAD65, the major autoantigen in IDDM, were constructed in pVEX1. These clones may become important tools to study the nature of GAD autoreactivity in IDDM. The clone pHICEO.9 was selected from the human insulinoma cDNA library by immunoscreening with total human insulinoma protein antibodies. This clone expresses the C-terminal fragment of human cholesterol esterase/lipase containing its antigenic determinant and can be used for blood lipase determination. Four clones containing cDNA inserts (0.47-1.42 kb) without any significant homologies to the known sequences in the Gene Bank were obtained by means of statistic selection.     

Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver lambda Zap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates beta-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.     

Structural and functional studies on the interaction of sodium dodecyl sulfate with beta-galactosidase

The effect of sodium dodecyl sulfate (SDS) on enzyme activity, electrophoretic behavior, and conformation of Escherichia coli beta-galactosidase is presented. Fourier-transform infrared spectroscopy (FT-IR), previously used to study the structure of native beta-galactosidase has been applied to examine the detergent effects on the enzyme. At 20 degrees C, the presence of 1% SDS does not cause appreciable changes in the secondary structure, and enzyme activity is preserved; however, 10% SDS produces complete enzyme inactivation and FT-IR spectroscopy indicates a concomitant change in conformation. Thermal denaturation of beta-galactosidase starts at approximately 53 degrees C in the absence and at approximately 46 degrees C in the presence of 1% SDS, indicating tertiary structure changes; also, a good correlation between structural (FT-IR) and functional (Arrhenius plots) data is observed. The secondary structure of thermally denatured beta-galactosidase contains mainly extended structures, and intermolecular interactions produce protein aggregation. In the presence of 10% SDS, however, the hydrophobic segments of the protein are stabilized by SDS into helical structures without protein aggregation. At 30 degrees C, in the presence of 1% SDS, two protein bands are resolved by gel electrophoresis, only one of them being active. A model for SDS-galactosidase interaction is proposed, according to which, at low surfactant concentrations, SDS molecules bind the outer surface of the protein, without affecting the protein core. Higher detergent concentrations produce a larger conformational change involving enzyme inactivation and increased accessibility of the solvent to the protein core. Increasing temperature in the presence of 10% SDS leads to a facilitated access of surfactant molecules to the inner protein regions and to an increase of the beta-galactosidase alpha-helical content.     

A new purification procedure for fumarase based of affinity chromatography. Isolation and characterization of pig-liver fumarase

A new procedure has been developed for the isolation of fumarase (EC 4.2.1.2). It is described for the purification of pigheart and liver enzyme. Pyromellitic acid has been covalently coupled to Sepharose-4B with diaminopropanol as spacer arr. When a dialysed 0.55 saturated ammonium sulphate precipitate is applied to the column, in Tris-acetate buffer, pH 7.3, fumarase remains quantitatively bound. It is eluted by competition, together with a few other proteins, by the natural product L-malate. Malate is removed from the eluate by dialysis. After this highly efficient purification step the enzyme is very easily crystallized. The final yield by 67% for both pig heart and liver preparations. The specific activity of fumarase purified from both tissues is found to be the same. Polyacrylamide gel electrophoresis in dodecylsulphate shows one single band corresponding with a subunit molecular weightof 48500. A single band is also obtained by electrophoresis in acid urea. This new procedure based on biospecific affinity chromatography allows a fast and easy preparation of gram quantities of fumarase.     

Effect of renal insufficiency on outcome following infrarenal aortic surgery

The molecular form and subcellular distribution of acid beta-galactosidase in cultured fibroblasts from patients with beta-galactosidase deficiency (GM1-gangliosidosis, Morquio B disease and galactosialidosis) were studied, using antibodies against three different forms of the human enzyme: a high-molecular-weight multienzymic complex, a recombinant 84-kDa precursor, and a 64-kDa tryptic product of the precursor. The mature enzyme from normal fibroblasts was immunoprecipitated by the anti-complex and anti-64-kDa protein antibodies, but not by the anti-84-kDa precursor one. immunofluorescence staining of normal fibroblasts revealed the granular (lysosomal) distribution with anti-64-kDa protein antibody and the perinuclear reticular distribution with anti-84-kDa precursor antibody, probably representing the Golgi apparatus. Both patterns were demonstrated in Morquio B disease, but the residual enzyme activity was exclusively due to the mature enzyme. In Type 1 galactosialidosis, most of the expressed enzyme was detected as the precursor form with a perinuclear reticular distribution. In type 2 galactosialidosis, more than half of the enzyme activity was due to the mature form with a lysosomal distribution. Fibroblasts from a patient with GM1 gangliosidosis, expressing no beta-galactosidase mRNA, did not react against either anti-64-kDa protein antibody or anti-84-kDa precursor antibody. The combined use of immunoprecipitation and immunostaining was useful for analysing the pathophysiology of the intracellular processing and transport of the mutant beta-galactosidase.     

O-ethyl 14C]phenacetin O-deethylase activity in human liver microsomes

The activity of human liver microsomal cytochrome P450 1A2 (CYP1A2) is readily estimated by following the O-deethylation of [O-ethyl 14C]phenacetin (PODase). The basis of the assay is the quantitative measurement of [14C]acetaldehyde, remaining in the supernatant of assay incubates, after extraction of unmetabolized [O-ethyl 14C]phenacetin with charcoal. In the presence of native human liver microsomes (K(m) = 54 +/- 27 microM; V(max) = 14 +/- 2.3 nmol/hr/mg; mean +/- SD; N = 3 different livers) and human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP1A2 (K(m) = 46 microM; V(max) = 55 nmol/hr/nmol CYP), PODase activity conformed to monophasic Michaelis-Menten kinetics. Furthermore, PODase activity in a panel of microsomes prepared from a series of human livers was significantly correlated (r = 0.91; p < 0.001; N = 11) with CYP1A2-selective 7-ethoxyresorufin O-deethylase activity, and was markedly inhibited (> or = 92%) by furafylline (FURA, IC50 = 0.4 microM) and 7,8-benzoflavone (ANF, IC50 = 0.1 microM), two well known CYP1A2 inhibitors. Inhibitors selective for other forms of CYP (e.g. CYP3A, CYP2C, CYP2D6, CYP2E1) elicited a marginal effect (< or = 17% inhibition) at relatively high concentrations (> or = 10.K(i)). It is concluded that the inhibition of human liver microsomal CYP1A2 activity can be readily determined by using a charcoal-based radiometric method employing [O-ethyl 14C]phenacetin as substrate.     

FT-IR measurement of mercaptoundecahydrododecaborate in human plasma

A simple and rapid method for the quantitative measurement of mercaptoundecahydrododecarborate (BSH), (which presently is one of the most useful agents for Boron Neutron Capture Therapy) in human plasma was developed by using Fourier transform infrared spectroscopy. Different spacer thicknesses of the liquid sampling cell were examined and the optimal results were obtained by the 0.05 mm spacer. The subtraction of water absorbance from sample spectra resolved a B-H band at 2493 cm-1. The quantitative measurement of BSH was carried out by integration of the B-H band in the wavenumber range of 2534-2440 cm-1. However, at the lower BSH concentration range, a visual inspection of the spectrum to determine the wavenumber range was necessary so as to avoid any negative areas to be integrated. The lower limit of detection of BSH in aqueous solution and human plasma was 5 micrograms ml-1 (about 2.5 ppm of boron).     

Kinetics of simultaneous saccharification and lactic acid fermentation processes

An inhibitory anti-peptide antibody was raised against a 21-amino acid peptide (VKRMKESRLEDTQKHRVDFLQ) corresponding to residues 253-273 of human cytochrome P450 3A4. High titer antibodies were produced by rabbits immunized with this peptide coupled to keyhole limpet hemocyanin, as judged by ELISA. Anti-peptide antibody recognized a single protein band in microsomes prepared from cells expressing recombinant human CYP3A4 in immunoblotting analysis. No immunodetectable proteins were found in microsomes containing other cytochrome P450 isoforms. In addition, the antibody did not recognize CYP3A5, a closely related isoform in the CYP3A family. In human liver microsomes, only one protein band which comigrated with human CYP3A4 was recognized by this antibody and the relative blotting intensity of this protein band correlated significantly with human CYP3A4-catalyzed testosterone 6 beta-hydroxylase activities (r = 0.96). More importantly, this antibody exhibited greater than 90-95% inhibition of testosterone 6 beta-hydroxylation, while other cytochrome P450-mediated reactions in human liver microsomes were not inhibited. Because of its specificity and inhibitory potency, this anti-peptide antibody should be a valuable tool in evaluating the role of CYP3A in mediating in vitro metabolism of therapeutic agents.