ABSTRACT
A thermolabile UPTG inhibitor protein (IP) was isolated and purified from a developing maize endosperm preparation. High homology of two internal peptides of IP with known plant sucrose synthase (SS) sequences suggested that IP might be related somehow with SS. IP and SS activities were found in the same preparation and showed thermolability between 60-65 degrees C. IP and SS activities presented the same ionic charge and molecular mass in native conditions (Mono Q and Superose-12 columns chromatographies). Western blot experiments with an anti-SS antibody as well as with an anti-IP antibody showed a single 80 kDa polypeptide band where IP and SS activities were present. Anti-SS antibody can neutralize SS as well as IP activities in a neutralization assay. It was found that in the maize mutant shrunken-1, lacking SS1 protein, the UPTG activity was not inhibited. Furthermore, the solubilized preparation of the sh1 endosperm is unable of inhibiting UPTG activity from potato tuber. The high correlation between IP and SS properties suggests that IP might be in fact a form of SS. Moreover, the relation between IP and the SS1 isoform is discussed. So, a new biological activity of SS is suggested.
Subject(s)
Enzyme Inhibitors/isolation & purification , Glucosyltransferases/antagonists & inhibitors , Glucosyltransferases/isolation & purification , Plant Proteins/isolation & purification , Zea mays/chemistry , Amino Acid Sequence , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glucosyltransferases/chemistry , Molecular Sequence Data , Neutralization Tests , Plant Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Protein complexes present different degrees of stability. We have previously described a glycoprotein from Bacillus thuringiensis that appeared as a multimer unable to be dissociated by the usual SDS-containing sample buffers of pH 6.8. In order to dissociate the complex, a SDS-containing sample buffer of pH 9 was described. In the present report three additional protein complexes with different degrees of stability and the effect of that dissociating sample buffer are described. The study of SDS critical micellar concentration values as a function of pH explains the improvement of dissociating properties at pH 9.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Sodium Dodecyl Sulfate , Bacterial Proteins/chemistry , Buffers , MicellesABSTRACT
One isoform of potato (Solanum tuberosum L., cv. Spunta), type L phosphorylase (EC 2.4.1.1), exhibiting primer independent activity appears to be tuber-specific. However, this activity can also be modulated by exogenous sucrose in storage as well as in non-storage organs. Primer independent phosphorylase (PIPh) activity in microtubers and shoots of in vitro plantlets was found to be much higher than in tubers and shoots of soil-grown plants. Detached leaves of soil-grown plants showed an increase in PIPh activity as well when incubated in sucrose-containing Murashige-Skoog (MS) medium. This increase was always accompanied by a rise in starch content. The presence of metabolizable carbohydrates in the growth or incubation medium are likely to be responsible for the observed rise in PIPh activity. In vitro microtubers and micropropagated plantlet organs (shoots and roots) exhibited a correlation between measurable PIPh activity and presence of enzyme protein, as judged by Western blot analysis using anti-potato tuber type L phosphorylase antibody. Therefore, in addition, to be developmentally regulated (tuber-specific accumulation), PIPh activity associated with the tuber type L isoform might be under a form of metabolic regulation.
Subject(s)
Isoenzymes/metabolism , Phosphorylases/metabolism , Solanum tuberosum/enzymology , Isoenzymes/analysis , Phosphorylases/analysis , Solanum tuberosum/growth & development , Starch/metabolism , Sucrose/metabolism , Tissue DistributionABSTRACT
UDP-glucose: protein transglucosylase (UPTG, EC 2.4.1.112) catalyzes the first step of protein-bound alpha-glucan synthesis in potato tuber and developing maize endosperm. The presence of a non-dialyzable, heat labile protein responsible for low levels of UPTG activity in developing maize endosperm was investigated. UPTG activity in 5-day old maize seedlings and potato tuber solubilized preparations was also reduced by the endosperm preparation. FPLC-Mono Q column chromatography of developing maize endosperm was effective in separating the inhibitor protein (IP) from UPTG. After gel filtration on Superose 12, IP yielded a major polypeptide of about 80 kDa on SDS-PAGE. IP was purified by gel filtration on Superose 12 and preparative SDS-PAGE, and specific antibodies were prepared. Polyclonal antibodies reacted specifically with an 80 kDa polypeptide of developing maize endosperm on Western blot. They also recognized a similar band in 5-day old maize seedlings, but not in potato tubers. The identification of a factor that regulates the level of UPTG activity in developing maize endosperm may help to elucidate the functional role of the enzyme in the initiation of starch synthesis during seed development.
Subject(s)
Enzyme Inhibitors/pharmacology , Glucosyltransferases/antagonists & inhibitors , Plant Proteins/pharmacology , Zea mays/enzymology , Enzyme Inhibitors/isolation & purification , Molecular Weight , Plant Proteins/isolation & purification , Solubility , Starch/biosynthesis , Zea mays/growth & development , Zea mays/metabolismABSTRACT
Two glycoproteins (205 and 72 kDa) were found in Bacillus thuringiensis sporangia. They were predominantly localized in the exosporium and/or the spore coat, although a small proportion was also found in membranes. A method for the dissociation of hydrophobic aggregates that resist the usual conditions of SDS-PAGE is described. Using this method we established that the 205 kDa glycoprotein is a multimer of the 72 kDa one. Deglycosylation of the 205 kDa and 72 kDa glycoproteins with trifluoromethanesulfonic acid yielded a 54 kDa polypeptide in both cases. At least three species of oligosaccharides were O-glycosidically linked to serines of the 54 kDa polypeptide chain. One of the oligosaccharides had N-acetylgalactosamine at the reducing end, rhamnose and a component not yet identified.
Subject(s)
Bacillus thuringiensis/chemistry , Bacterial Proteins/chemistry , Glycoproteins/chemistry , Oligosaccharides/chemistry , Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , Chromatography, Gel , Chromatography, Ion Exchange , Chromatography, Paper , Electrophoresis, Polyacrylamide Gel , Glucose/metabolism , Glycoproteins/biosynthesis , Glycoproteins/isolation & purification , Hydrogen-Ion Concentration , Pronase/metabolism , Protein Conformation , Serine/metabolism , Spores, Bacterial , Time FactorsABSTRACT
Potato (Solanum tuberosum L.) tuber UDP-glucose:protein transglucosylase (UPTG) (EC 2.4.1.112) is involved in the first of a two-step mechanism proposed for protein-bound alpha-glucan synthesis by catalyzing the covalent attachment of a single glucose residue to an acceptor protein. The resulting glucosylated 38-kilodalton polypeptide would then serve as a primer for enzymic glucan chain elongation during the second step. In the present report, we describe the fast protein liquid chromatography purification of UPTG from a membrane pellet of potato tuber. An apparently close association of UPTG, phosphorylase, and starch synthase was observed under native conditions during different purification steps. Enrichment of a 38-kilodalton polypeptide was found throughout enzyme purification. It is now shown that the purified UPTG, with an apparent molecular mass of 38 kilodaltons, undergoes self-glucosylation in a UDP-glucose- and Mn(2+)-dependent reaction. Therefore, it is concluded that UPTG is the enzyme and at the same time the priming protein required for the biogenesis of protein-bound alpha-glucan in potato tuber.