[Expression and purification of human sucrase protein in E.coli].

OBJECTIVE: To construct prokaryotic expression vector pET-28a(+)-human sucrase (hSUC) and express hSUC fusion protein in E.coli. METHODS: The hSUC gene fragment was amplified by reverse transciption PCR (RT-PCR) and cloned into pET-28a(+) vector to construct the prokaryotic expression vector pET-28a(+)-hSUC. The recombinant plasmid was then transformed into E.coli BL21. Hisdidine (His)-tagged fusion proteins were induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by nitrilotriacetic acid (Ni-NTA) agarose resin. The purified fusion proteins were identified by SDS-PAGE and Western blotting. RESULTS: RT-PCR showed that sub-clone of hSUC was about 1482 bp. The recombinant plasmid was correctly constructed as demonstrated by sequencing and restriction enzyme analysis. The molecular mass of the fusion protein was about 61 240. Western blotting showed that the fusion proteins bound specifically to hSUC antibody. CONCLUSION: The hSUC protein has been successfully expressed and purified in E.coli.

Purification and characterization of fructan and fructansucrase from Lactobacillus fermentum AKJ15 isolated from Kodo ko jaanr, a fermented beverage from north-eastern Himalayas.

Fructansucrase and fructan produced from Lactobacillus fermentum AKJ15 were isolated from seeds of Kodo ko jaanr, a fermented mild-alcoholic beverage prepared in North East India. The strain was identified by 16S rRNA gene sequence analysis and biochemical characterization. The strain displayed maximum fructansucrase activity of 4.3 U/ml (1.02 U/mg) at 28°C at 180 rpm. The enzyme purified by polyethylene glycol-400 gave specific activity of 5 U/mg and showed 90 kDa band on non-denaturing Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE). The purified enzyme confirmed the presence of fructan by periodic acid Schiff's staining which showed magenta colour bands with both sucrose and raffinose. The strain produced 10.2 mg/ml fructan in broth under optimized culture conditions. The purified fructansucrase displayed V(max) of 5.42 U/mg and K(m) of 16.65 mM. The enzyme showed maximum activity at 30°C and at pH 5. The structure of fructan was analysed by (1)H and (13)C NMR spectra confirming ß-(2-1) and ß-(2-6) linkages.

Regulation of cellobiase secretion in Termitomyces clypeatus by co-aggregation with sucrase.

Regulated secretory proteins are sorted via selective co-aggregation in eukaryotes. Cellobiase (C) of the filamentous fungus Termitomyces clypeatus remained co-aggregated with sucrase (S), and only one isoform of each of the enzymes was present in intra- and extracellular extracts. Kinetics of secretion of sucrase increased in vivo and in vitro in secreting (Sc) medium and decreased under non-secreting (NSc) conditions similar to those observed for cellobiase. In the Sc condition, total enzyme production and activity ratios of cellobiase and sucrase (C/S) in cell-bound, extra- and intracellular preparations increased with time and were significantly higher from those obtained in non-secretory media. It was concluded that secretion of sucrase in culture medium is under same cellular regulation as that of cellobiase, and sucrase is involved in regulating extracellular release of cellobiase through co-aggregation in the fungus.

Regulation (alteration) of activity and conformation of sucrase by coaggregation with cellobiase in culture medium of Termitomyces clypeatus.

Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS-PAGE and HPGPLC. K(m) and V(max) of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 degrees C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.

An aminopeptidase N deficiency in dog small intestine.

This study has identified a naturally occurring, specific deficiency of a brush border aminopeptidase N (ApN) in the small intestines of five clinically healthy dogs. ApN activity in mucosal homogenates of dog small intestine was reduced significantly in deficient animals (13.4 (1.1) nmol min-1 mg-1 protein, n = 5, P < 0.002) compared to healthy control dogs (95.1 (6.7), n = 22). Alkaline phosphatase, gamma-glutamyl transferase, zinc-resistant alpha-glucosidase, maltase, sucrase and lactase in the ApN deficient dogs exhibited comparable activities to those in the control dogs. Microvillar membranes were analysed by one- and two-dimensional electrophoresis. ApN was represented by a single 145kDa band in all control dogs, identified by immunoblotting and immunoprecipitation. Protein maps from deficient dogs were normal apart from the virtual absence of an ApN spot and there were no apparent abnormalities in the glycosylation of microvillar proteins. The findings suggest that intestinal ApN deficiency in these dogs is a primary lesion involving diminished expression of an otherwise normal enzyme protein.

The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA.

The complete nucleotide sequence of the surA gene, encoding a sucrase from Bacillus stearothermophilus NUB36, was determined. surA was composed of 1338 bp and encoded 445 amino acid residues. The deduced polypeptide of M(r) 51519 showed strong sequence similarity to sucrose and sucrose phosphate hydrolases from Bacillus subtilis, Klebsiella pneumoniae and Vibrio alginolyticus, and contained the 'sucrose box' residues thought to be important for catalysis of the transfer of fructose from sucrose. The enzyme was partially purified using affinity chromotography from extracts of Escherichia coli containing the cloned surA. SurA displayed an optimum temperature for sucrose hydrolysis of 55 degrees C and high stability. The M(r) of SurA determined by gel filtration was 105,000, which suggested that the active form of the enzyme is a dimer. SurA exhibited an apparent Km of 40 mM for sucrose but, unlike the homologous B. subtilis enzyme, had no detectable sucrose phosphate hydrolase activity.

Glycosidases in Leishmania and their importance for Leishmania in phlebotomine sandflies with special reference to purification and characterization of a sucrase.

Culture forms of Leishmania (Leishmania) amazonensis (IFLA/BR/67/PH8) produce an extracellular enzyme that hydrolyzes sucrose molecules into their component monosaccharides. This is important because phlebotomine sand flies, the invertebrate hosts of Leishmania, ingest plant sap or aphid and coccid honeydew rich in sucrose between blood meals and Leishmania promastigotes cannot uptake sucrose. The sucrase was purified and characterized; its molecular weight, estimated by gel filtration chromatography and SDS-PAGE electrophoresis, was about 73 kDa. K(m) and V(max) measured with sucrose as substrate were respectively 4.4 mM and 6.9 mumole glucose.min-1 (mg sucrase)-1, with maximum pH activity at pH 5.5. A series of natural and p-nitrophenyl-derived substrates were assayed, characterizing the enzyme as a highly specific beta-D-fructofuranoside fructohydrolase. When 11 species of Leishmania and 7 genera of trypanosomatids were screened, only the species of the genus Trypanosoma did not produce an enzyme with saccharolytic activity. These data are in agreement with the fact that the latter vectors do not acquire sucrose or raffinose in their meals. Searching for glycolytic enzymes other than sucrase, we found an N-acetyl-beta-D-galactosaminolytic activity. This N-acetyl-galactosaminidase, here described for the first time, might have a role in peritrophic membrane disruption. The importance of sucrase and N-acetyl-beta-D-galactosaminidase in the Leishmania life cycle is discussed.

Characterisation of an acid trehalase of Saccharomyces cerevisiae present in trehalase-sucrase aggregate.

An acid trehalase-sucrase aggregate was purified (by 780-fold) from Saccharomyces cerevisiae, following conventional protein purification techniques, to an apparent yield of 18.5%. The aggregate was electrophoretically homogeneous but contained 175, 90, 68, 60, 40 molar mass (kDa) bands on SDS-electrophoresis. The purified aggregate had a specific activity (acid trehalase) of 22 U/mg; a Km value of 5.0 mM but contained 3-times more sucrase activity. Only sucrose and trehalose were hydrolysed by this aggregate and both activities were inhibited by acetate or phosphate. Temperature and pH optima for trehalose hydrolysis appeared to be 40-45 degrees C and 5.0, respectively. The purified aggregate appeared to be disaggregating spontaneously resulting in inactivation of both enzymes, which was enhanced either at pH 3.5 or at pH 7.0. Separation of acid trehalase from the aggregate by hydrophobic interaction chromatography resulted in inactivation. Rechromatography (HPGPLC) of the purified aggregate also gave disaggregation as well as inactivation of both enzymes. Disaggregated acid trehalase and sucrase contained 20-fold and 13-fold lower specific activities, respectively, and appeared to be unstable. Based on these observations we suggest that acid trehalase is stabilised by aggregation with sucrase.

Feeding status affects in vivo prosucrase.isomaltase processing in rat jejunum.

Fed and fasted (18 h) adult male rats received a primed constant infusion of [3H]leucine for 15 to 180 min. Prosucrase-isomaltase (pro-SI) and sucrase were isolated from mixed jejunal mucosal membranes by immunoprecipitation and separated from one another by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The rate at which pro-SI was processed to sucrase was calculated on the assumption that the steady-state specific radioactivity of leucine in pro-SI defined the pool of amino acids used in the formation of brush border sucrase. At isotopic steady state, pro-SI achieved a specific radioactivity that was higher than that of mucosal free leucine in both feeding groups. The relationship between the isotopic equilibrium of the free amino acid pools and pro-SI was sensitive to feeding status; the specific radioactivity of pro-SI was 25 and 55% (P less than 0.05) of the blood specific radioactivity in fed and fasted animals, respectively. The fractional rate of pro-SI processing tended to be higher (P less than 0.07) in fed (407%/d) than in fasted animals (274%/d). We conclude that the general mucosal free amino acid pool is not the amino acid pool from which pro-SI is synthesized and that the rate of pro-SI processing from the endoplasmic reticulum-Golgi membranes to the brush border membrane is sensitive to the feeding status of the animal.

Detection and characterization of sucrase-isomaltase in adult human colon and in colonic polyps.

A panel of monoclonal antibodies specific for sucrase-isomaltase, but differing in their ability to stain the proliferative crypt cells in human jejunum, was used to investigate expression of this enzyme in adult human colon and colonic tumors. Immunofluorescence staining on cryostat sections demonstrated the presence of sucrase-isomaltase in the apical region of normal colonic crypt cells but not on surface epithelium. Colonic sucrase-isomaltase was purified by immunoprecipitation with selected monoclonal antibodies and identified predominantly as high-mannose and complex glycosylated single-chain precursors endowed with relatively low levels of enzyme activities. Most polyps examined (10/16) were also found to express significant amounts of sucrase-isomaltase. In contrast, only 3 of 45 adenocarcinomas were positive by immunofluorescence staining; no correlation was found between enzyme expression and tumor classification either by "Dukes" stage or degree of histological differentiation. These results demonstrate that colonic crypt cells and some benign tumor cells synthesize and express at their cell surface a form of sucrase-isomaltase immunologically distinct from that present in the brush borders of small intestinal villose cells.

pH-dependent inhibitory effects of tris and lithium ion on intestinal brush-border sucrase.

Tris and two of its hydroxylated amine analogs were examined in a metal-free, universal n-butylamine buffer, for their interaction with intestinal brush border sucrase. Our recent three-proton-families model (Vasseur, van Melle, Frangne and Alvarado (1988) Biochem. J., 251, 667-675) has provided the sucrase pK values necessary to interpret the present work. At pH 5.2, 2-amino-2-methyl-l-propanol (PM) causes activation whereas Tris has a concentration-dependent biphasic effect, first causing activation, then fully competitive inhibition. The amine species causing activation is the protonated, cationic form. The difference between the two amines is related to the fact that Tris has a much lower pKa value than PM (respectively, 8.2 and 9.8). Even at pH 5.2, Tris (but not PM) exists as a significant proportion of the free base which, by inhibiting the enzyme fully competitively, overshadows the activating effect of the cationic, protonated amine. Above pH 6.8, both Tris and PM act as fully competitive inhibitors. These inhibitions increase monotonically between pH 6.5 and 8.0 but, above pH 8, inhibition by 2.5 mM Tris tends to diminish whereas inhibition by 40 mM PM increases abruptly to be essentially complete at pH 9.3 and above. As pH increases from 7.6 to 9.0, the apparent affinity of the free amine bases decreases whereas that of the cationic, protonated amines, increases. In this way, the protonated amines replace their corresponding free bases as the most potent inhibitors at high pH. The pH-dependent inhibition by 300 mM Li+ is essentially complete at pH 8, independent of the presence or absence of either 2.5 mM Tris or 40 mM PM. Even at pH 7.6, an excess (300 mM) of Li+ causes significant increases in the apparent Ki value of each Tris, PD (2-amino-2-methyl-1-3-propanediol) and PM, suggesting the possibility of a relation between the effects of Li+ and those of the hydroxylated amines which in fact are mutually exclusive inhibitors. The inhibitory results are interpreted in terms of a mechanistic model in which the free bases bind at two distinct sites in the enzyme's active center. Binding at the glucosyl sub-site occurs through the amine's free hydroxyl groups. This positioning facilitates the interaction between the lone electron pair of the deprotonated amino group with a proton donor in the enzyme's active center, characterized by a pK0 around 8.1. When this same group deprotonates, then the protonated amines acting as proton donors replace the free bases as the species giving fully competitive inhibition of sucrase.

Presence of sucrase in the yolk sac of the chick.

The presence of sucrase in the yolk sac of the chick was studied biochemically and immunologically. The sucrase was partially purified from the yolk sac of hatched chicks and was compared with the sucrase purified from the small intestine. Immunodiffusion with antiserum against intestinal sucrase and characterization of the activity revealed that the two enzymes were almost identical. However, the size of the yolk sac sucrase was found to be slightly smaller than that of the intestinal enzyme by chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis. Immunocytochemical studies showed that the sucrase was located on the free surface of yolk sac endodermal cells, but the sucrase may also be present in the cytoplasm.

Origin and function of the multiple extracellular glucosyltransferase species from cultures of a serotype c strain of Streptococcus mutans.

Two methods were used to purify the bifunctional extracellular enzyme sucrose: (1-6)- and (1-3)-alpha-D-glucan-6-alpha-D-glucosyltransferase (EC 2.4.1.5; dextransucrase) from continuous cultures of a serotype c strain of Streptococcus mutans. The first method, based on a previously published report, involved Sepharose 6B gel filtration and DEAE cellulose anion exchange chromatography. This resulted in a dextransucrase preparation with an apparent molecular mass of 162 kDa and a specific activity of 125 mg of glucan formed from sucrose h-1 (mg of protein)-1, at 37 degrees C. It was almost homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ratio of carbohydrate to protein was 0.14 and the recovery was 14% relative to the total glucosyltransferase activity in the original culture fluid. In the subsequently preferred method, hydroxyapatite-Ultrogel was used to purify dextransucrase with a 24% yield. The specific activity, 197 mg of glucan formed h-1 (mg of protein)-1, was the highest yet reported and this preparation contained less than 0.5 glucose-equivalent per subunit of molecular mass 162 kDa. Dextransucrase is therefore not a glycoprotein. Exogenous dextran stimulated activity, but was not essential for activity. The purified protein slowly degraded to multiple lower molecular mass forms during storage at 4 degrees C and 87% of the activity was lost after 20 days. The molecular mass of the most prominent, active degradation product was 140 kDa, similar to that of one of the multiple forms of dextransucrase detected in other laboratories. Preparations in which either the 140-kDa or the 162-kDa species predominated catalyzed the synthesis of a water-soluble glucan with sucrose alone, but catalyzed that of an insoluble glucan with sucrose and a high concentration of either (NH4)2SO4 or polyethylene glycol. The water-insoluble glucan was shown to lack sequences of 1,3-alpha-linked glycosyl residues typical of the insoluble glucan, mutan, which has been implicated in dental caries. We conclude that mutan is synthesized by the concerted action of two independent glucosyltransferases rather than by interconvertible forms of a single enzyme, as was proposed previously.

Purification and partial characterization of chick intestinal sucrase.

The sucrase was purified from the small intestinal mucosa of the adult chick. Purification procedure involved solubilization with papain, ethanol precipitation, chromatography on Sephadex G-200 and DEAE-Sephadex. Several characters of the chick intestinal sucrase resembled those of the intestinal sucrase-isomaltase complex of some mammals (rabbit, rat and human).

Isolation and localization of plasma membrane-bound invertase in yeast (Saccharomyces cerevisiae).

Latex spheres of 60 nm diameter (synthesized by aqueous emulsion copolymerization of methacrylate derivatives according to [22]) were coated with bovine serum albumin (BSA) and concanavalin A. By virtue of their size and their high density (1.32-1.35 g/ml) they are well suited as scanning electron microscopy markers and as affinity density perturbation reagents. Yeast protoplasts could be labeled with these spheres and the amount of binding depended upon incubation time and temperature. Isolated and solubilized yeast plasma membranes were incubated with these spheres and by density gradient centrifugation the membrane glycoproteins could be separated from the other proteins by the method of affinity density perturbation. Since the yeast plasma membrane glycoproteins exhibit invertase activity [1, 19] the activity of the different fractions was either detected on gels by staining for invertase activity or measured in vitro and quantified; a 6 to 7fold purification of the enzyme was achieved. Protoplasts labeled with antibodies directed against these glycoproteins exhibited a distribution of ferritin marker molecules that was very similar to that of the intramembranous particles. Antibodies against extracellular invertase cross reacted with the plasma membrane of glycoproteins and showed the same distribution of markers as the antibodies against the glycoproteins. It can therefore be concluded that the yeast plasma membrane glycoproteins exhibit invertase activity and that they are associated with the intramembranous particles.

[Purification and characterization of beta-fructofuranosidase from yeast Saccharomyces cerevisiae]. / Vydelenie i izuchenie svoistv beta-fruktozuranozidazy iz drozhzhei Saccharomyces cerevisiae.

Intracellular invertase was isolated from the yeast Saccharomyces cerevisiae, race XI, and purified by ion-exchange chromatography on DEAE-cellulose and gel-filtration on Sephadex G-200. The effect of pH, temperature, metal ions, thiolic agents, and EDTA on the enzyme activity and stability was investigated. The enzyme was estimated to have a molecular weight of 270 000 and a carbohydrate content of 20--30%. By disc-electrophoresis and isoelectric focusing the highly purified enzyme was found to be heterogenous. Its molecular forms had isoelectric points at 3.0, 4.0, 4.5, and 4.9.

Partial purification, characterization and localization of the membrane-associated invertase of yeast.

The membrane-associated isozyme of invertase (beta-D-fructofuranoside fructo-hydrolase, EC 3.2.1.26) -- precursor of the external glycoprotein invertase (Babczinski, P. and Tanner, W. (1978) Biochim. Biophys. Acta 538, 426-434) - has been purified 60-fold from deoxycholate extracts of derepressed yeast cells. The partially purified enzyme exhibits considerable stability as a salt-free lyophilized powder. Its molecular weight, in this precursor form, has been determined by by sodium dodecyl sulphate (SDS) gel electrophoresis to be 180 000 daltons. This correlates well with the presence of only the inner core carbohydrate parts of the external invertase. The enzyme can be split completely by treatment with endo-beta-N-acetyl-glucosaminidase H from Streptomyces griseus, demonstrating the presence of a di-N-acetylchitobiosyl-asparagine linkage. The proteinaceous split product is still active and has a molecular weight of approx. 120 000. The enzyme cannot be transferred into a supernatant fraction upon osmotic shock treatment of yeast membrane vesicles, indicating that it is strictly membrane-bound. After separation of yeast membranes on a sucrose density gradient, precursor invertase is predominantly associated with two gradient membrane fractions which most probably represent rough and smooth endoplasmic reticulum.