Greetje H. Vos-Scheperkeuter

Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.)

SummaryAn amylose-free potato mutant was isolated after screening 12,000 minitubers. These minitubers had been induced on stem segments of adventitious shoots, which had been regenerated on leaf explants of a monoploid potato clone after Röntgen-irradiation. The mutant character is also expressed in subterranean tubers and in microspores. Starch granules from the mutant showed a strongly reduced activity of the granule bound starch synthase and loss of the major 60 kd protein from the starch granules.

MECHANISM OF SUGAR-TRANSPORT THROUGH THE SUGAR-SPECIFIC LAMB CHANNEL OF ESCHERICHIA-COLI OUTER-MEMBRANE

SummaryLipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel ofEscherichia coli outer membrane. Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior. The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase. Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars. The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel. These carbonyl groups may also be involved in the sugar binding via hydrogen bonds. The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M. Luckey and H. Nikaido (Proc. Natl. Acad. Sci. USA77:165–171 (1980a)) and the stability constants for the sugar binding given in this study.

Assembly pathway of newly synthesized LamB protein an outer membrane protein of Escherichia coli K-12

The assembly of newly induced LamB protein (phage lambda receptor) was investigated in an operon fusion strain of Escherichia coli, in which the lamB gene is expressed under lac promoter control. The induction kinetics both for total cellular and for cell surface-exposed LamB protein were studied by immunochemical detection methods, using two distinct antisera directed against detergent-solubilized LamB trimers and completely denatured LamB monomers, respectively. Anti-trimer antibodies recognized both monomers and trimers, whereas anti-monomer antibodies only reacted with monomers. Provided appropriate solubilization conditions were used, both antisera were able to immunoprecipitate intracellular mature LamB protein quantitatively. Following induction, the first LamB antigenic determinants were detected after 60 to 80 seconds; detection of the newly synthesized protein by anti-monomer antibodies slightly preceded that by anti-trimer antibodies, a finding that could be partly explained by the observation that anti-monomer antibodies recognized a larger fraction of nascent LamB than did anti-trimer antibodies. Exposure of antigenic determinants at the cell surface was delayed for 30 to 50 seconds with respect to their synthesis. Therefore, either translocation or conformational changes must be rate-limiting in the series of processes that eventually convert the newly synthesized protein into its mature outer membrane state. LamB protein was found to occur in at least three clearly distinguishable states. State I is the LamB monomer, state II corresponds to a metastable trimer that dissociates in sodium dodecyl sulphate above 60 degrees C, and state III is the state LamB trimer that dissociates in sodium dodecyl sulphate only at temperatures above 90 degrees C. The chase kinetics of these states showed that conversion of newly synthesized LamB monomers to stable LamB trimers occurred in two stages: state I monomers were chased into metastable state II trimers rapidly (t 1/2 = 20 s), whereas stabilization of state II trimers to state III trimers was a relatively slow (t 1/2 = 5.7 min) process. Based on our results, a timing sequence in the assembly of outer membrane LamB protein is proposed.

Histochemical analysis of the role of class I and class II Clostridium histolyticum collagenase in the degradation of rat pancreatic extracellular matrix for islet isolation

To understand why class II Clostridium histolyticum collagenase is much more effective than class I in the isolation of rat pancreatic islets, we analyzed the role of these collagenases in pancreatic tissue dissociation. Crude collagenase was purified and then fractionated into class I and II with different enzyme activities and protein compositions. Pancreatic tissue was incubated with either class I, class II, or class I + II, with or without added protease, under conditions that eliminated endogenous proteolytic activity. The degradation of pancreatic extracellular matrix was monitored by selective histochemical staining of tissue samples. Class I and II showed similar capacities to degrade glycoproteins and degraded about one-third of the glycoproteins during 120 min of incubation. The degradation of collagens by class I and II was relatively more effective, 80 to 95% of the collagens being removed in 120 min, and also class dependent. Both in the presence and absence of protease, class II was more effective at degrading collagens than class I, but this difference in efficacy was less apparent than with islet isolation. Class I + II degraded collagens faster and more complete than did the individual classes, indicating a synergistic effect of class I and II. Evaluation of collagen degradation at various pancreatic locations did not show a selective degradation of collagens by any of the collagenase classes. The present data offer a partial explanation for the major role of class II in islet isolation.

Biosynthesis of starch; identification of potato starch enzymes

Abstract Two important starch enzymes, granule-bound starch synthase and branching enzyme, were purified from potato tubers and characterized by immunological comparison with the corresponding enzymes of other plants. Granule-bound starch synthase was identified as a 60-kd protein homologous to the corresponding enzymes of maize and amaranth; the enzyme was missing in amylose-free potato starch granules. Branching enzyme of potato tubers was purified as a single protein species of 79 kd which appeared to be homologous to maize branching enzyme I, but much less to branching enzymes IIa and IIb.