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Query: EC:3.2.1.26 (invertase)
 4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plant invertases play important roles in sucrose metabolism. Cell wall invertase was reported to participate in phloem loading and unloading. Soluble invertases would be involved in hexose level regulation in mature tissues and in stored sucrose utilization within vacuoles. Invertase inhibitory proteins were described as one of the possible mechanisms for invertase activity regulation in some plant species; nevertheless, these proteins were found only in sink tissues, suggesting that this mechanism would not be relevant in the sucrose turnover of leaves. This report describes the purification of invertase from Pteris deflexa fronds and the occurrence of an invertase inhibitory protein in this fern organ, as well as its purification and invertase-inhibitor interactions. The Mr of the invertase and of its inhibitory protein were 90,000 and 18,000, respectively. SDS-PAGE in the presence of 2-mercaptoetanol gave two subunits for the enzyme (Mr=66,000 and 30,000) and only one for the inhibitor. The inhibitor protein is a glycoprotein (12% w/w of neutral sugars) that did not show agglutinating activity like some others, and also showed a high heat stability at pH 5.0. The optimum pH of invertase activity is 5.0, while invertase inhibitory protein caused maximal inhibition at the same pH value. Invertase-inhibitor complex formation occurs in an immediate manner and a protease activity was discarded. The inhibition is non-competitive (Ki=1.5 x 10(-6) M) without interactions among the binding sites. The complex is slightly dissociable and sucrose was able to partially reduce the inhibitory effect. Up to the present, invertase inhibitory proteins have been found solely in heterotrophic tissues. In this work we demonstrate that this protein is also present in an autotrophic tissue of a lower vascular plant.
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PMID:An invertase inhibitory protein from Pteris deflexa link fronds. 1216 91

The extracellular cellobiase (EC 3.2.1.21) of Termitomyces clypeatus separated in two protein fractions when culture filtrate or ammonium sulfate precipitated proteins were chromatographed on BioGel P-200 column. During purification of cellobiase (CBS) from the lower molar mass (LMM) protein fraction, the enzyme behaved like a low molecular weight multimeric protein. The purified enzyme gave a single 56 kDa band in SDS-PAGE but ladderlike bands (14, 28, 42, and 56 kDa) on denaturation by reducing-SDS and urea. The protein, however, dissociated on dilution and protomeric (14 kDa) and multimeric forms (28 and 60 kDa) were eluted separately during HPGPLC. Specific activity of CBS gradually decreased as the molar mass of the enzyme was lowered in different eluted peaks. Protein present in all CBS pool fractions had the same amino acid composition and all displayed the same, single protein peak in reverse-phase HPLC and 56 kDa band in SDS-PAGE. Thus, T. clypeatus CBS was a multimeric 14 kDa protein that was optimally active as a tetramer. CBS purified from the higher molar mass fraction (HMM) as a SDS-PAGE homogeneous 110-kDa protein did not dissociate on dilution or by SDS-urea. The purified protein was a protein aggregate as CBS consistently contained 20 +/- 5% sucrase (SUC) Units in the preparation. The aggregate resolved during reverse-phase chromatography on a C(4) column, and an additional protein peak other than CBS was detected. The aggregated CBS had a higher temperature optimum and was more stable toward thermal and chemical denaturations than SUC-free CBS. Increase of stability and catalytic activity of CBS by aggregation with SUC was much higher than those by the multimerization of CBS itself. All of these observations for the first time suggested that the heterologous protein-protein aggregation, observed for a long time for fungal enzymes, might have a significant role in modulating physicochemical properties of the extracellular enzyme.
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PMID:Stabilization and improvement of catalytic activity of a low molar mass cellobiase by cellobiase-sucrase aggregation in the culture filtrate of Termitomyces clypeatus. 1246 58

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.
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PMID:Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit. 1271 Nov 32

Acetyl esterase (AE) activity present in the culture filtrate of Termitomyces clypeatus was separated into lower molar mass (LMM) and higher molar mass (HMM) protein fractions during BioGel P-200 gel chromatography. AE was purified as a 30 kDa nonglycosylated protein from LMM fractions by CM-Sepharose ion exchange chromatography and HPGPLC. Although the HMM fraction had a number of enzyme activities (sucrase, beta-xylosidase, beta-glucosidase, and alpha-L-arabinofuranosidase) other than AE, protein present in the fraction was eluted as a single protein peak in HPGPLC and gave a single band in native PAGE. The fraction, subsequently purified by DEAE-Sephadex chromatography, was a SDS-PAGE homogeneous 80 kDa glycoprotein, but with both AE and cellobiase activities. The aggregate dissociated during ConA-Sepharose chromatography and 30 kDa AE and 56 kDa glycosylated cellobiase were purified separately. The dissociation caused significant loss of cellobiase activity but not that of AE. AE purified from both HMM and LMM fractions was characterized to be the same enzyme in terms of molar masses, pI (7.3), and other physicochemical properties. AE as an aggregate with cellobiase showed higher thermostability, temperature optimum, and resistance toward chemical denaturants than those of purified AE. Compared to cellobiase purified earlier from the same fungus, the enzyme present with AE in the aggregate also showed higher catalytic activity, thermostability, and temperature optimum. The study indicated that the formation of such SDS-resistant enzyme aggregate was associated with significant changes in the physicochemical properties of the enzymes, mainly toward improvement of rigidity of enzymes, and sometimes with the improvement of catalytic activity.
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PMID:Protein-protein interaction conferring stability to an extracellular acetyl (xylan) esterase produced by Termitomyces clypeatus. 1279 Jun 30

Attempts to purify the inhibitor of pectin methylesterase (PMEI) from the soluble extract of ripe apricot (Prunus armeniaca) fruit led to isolation of a protein (Pa-INH) similar to PMEI, but having invertase inhibitory activity against vacuolar invertase from tomato. The molecular charge, the native and SDS-PAGE molecular weights were similar to those of PMEI. Partial amino acid sequence indicated a high level of identity with invertase inhibitors and a significant identity with PMEI. Circular dichroism analysis showed a mainly alpha-helix secondary structure for both the inhibitors and a higher thermostability of Pa-INH. Four Cys residues forming disulfide bridges in PMEI were conserved in Pa-INH. Similarly to PMEI, these residues were linked by disulfide bridges (first to second and third to fourth). The free Cys139 of PMEI is substituted by Ala in Pa-INH. The results reported in this study suggest a common structural arrangement of the two inhibitors.
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PMID:The plant invertase inhibitor shares structural properties and disulfide bridges arrangement with the pectin methylesterase inhibitor. 1367

Two proteinaceous invertase inhibitors, designated ITI-L and ITI-R, were purified to electrophoretic homogeneity. ITI-L was purified from acetone powder of sweet potato leaves through sequential steps entailing buffer extraction, acid treatment, DEAE-Sephacel ion-exchange chromatography, and Sephacryl S-100 gel filtration. ITI-R was purified from sweet potato tuberous roots by sequentially applying buffer extraction, Con A-Sepharose affinity chromatography, DEAE-Sephacel ion-exchange chromatography, Sephacryl S-200, and Superose 12 gel filtration. The optimal pHs for interaction between ITI-L and ITI-R and acid invertase from sweet potato leaves were 5.5 and 5.0, respectively. The molecular masses of ITI-L and ITI-R were 10 and 22 kDa, respectively, as estimated by both gel filtration and SDS-PAGE. Both inhibitors were thermostable (90% of the activity remained after incubation at 100 degrees C for 20 min), and Western blotting showed them to be immunologically related.
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PMID:Invertase inhibitors from sweet potato (Ipomoea batatas): purification and biochemical characterization. 1470 16

The present study analyzed the existence of carbohydrases in camel pancreas compared to some other ruminants. Disaccharidases (maltase, cellobiase, lactase, trehalase and sucrase), glucoamylase and alpha-amylase were detected in pancreas of camel, sheep, cow and buffalo. Enzyme levels in sheep were lower than in the other ruminants. The highest level was detected for alpha-amylase (EC 3.2.1.2). Moderate activity levels were detected for glucoamylase (EC 3.2.1.3) and maltase (EC 3.2.1.20), while other disaccharidases showed very low activity. The results suggested that, in addition to alpha-amylase, glucoamylase and maltase may be synthesized and secreted from pancreas to the small intestine in ruminants. Camel pancreatic glucoamylase was purified and characterized. The purification procedure included glycogen precipitation and chromatography on DEAE-Sepharose and Sepharose 6B. The molecular mass was 58 kDa for native and denatured enzyme using gel filtration and SDS-PAGE, respectively. The enzyme had a pH optimum at 5.5 and a Km of 10 mg starch/mL with more affinity toward potato soluble starch than the other carbohydrates. Glucoamylase had a temperature optimum at 50 degrees C with heat stability up to 30 degrees C. The effect of different cations and inhibitors was examined. The camel pancreatic glucoamylase may possess an essential thiol.
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PMID:Carbohydrases in camel (Camelus dromedarius) pancreas. Purification and characterization of glucoamylase. 1562 12

An endoinulinase produced by Chaetomium sp. C34 was purified to electrophoretic homogeneity, with recovery of 7.7% activity and purification factor of 30.8 fold by five steps including ammonium sulfate precipitation, DEAE-cellulose, Q-sepharose Fast Flow, Sephacryl S-200 and Pre-Packed Hydrophobic Column. Its subunit molecular weight was estimated to be about 66kD by SDS-PAGE. The optimum temperature and pH of the enzyme activity were 50 approximately 55 degrees C and 6.0 respectively. The K(m) and V(max) values for inulin were 0.199 mmol/L and 115 micromol/(mg x min) respectively. Cu2+ completely inhibited inulinase activity. An appreciable loss of activity was observed in presence of NBS, Mn2+, Zn2+, Fe2+ and EDTA. A ratio of inulinase activity to invertase activity (I/S) of 20 was found in purified inulinase. The endoinulinase hydrolyzed inulin and liberated inulooligosaccharides. But it lacked activity toward melezitose or raffinose.
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PMID:[Purification and properties of endoinulinase from Chaetomium sp]. 1611 Sep 61

Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol that includes sodium carbonate to prevent cytoplasmic contamination. These proteins were analyzed by SDS-PAGE, trypsin digestion and LC-MS/MS for peptide identification. Elongation factors, mitochondrial membrane proteins and a mannosyltransferase involved in cell wall mannan biosynthesis were also identified in this fraction.
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PMID:Genetic and proteomic evidences support the localization of yeast enolase in the cell surface. 1654 86

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation.
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PMID:Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release. 1657 Mar 16


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