Bruce P. Wasserman

Isolation and characterization of the zSSIIa and zSSIIb starch synthase cDNA clones from maize endosperm

Two starch synthase clones, zSSIIa and zSSIIb, were isolated from a cDNA library constructed from W64A maize endosperm. zSSIIa and zSSIIb are 3124 and 2480 bp in length, and contain open reading frames of 732 and 698 amino acid residues, respectively. The deduced amino acid sequences of the two clones share 58.1% sequence identity. Amino acid sequence identity between the zSSIIa and zSSIIb clones and the starch synthase II clones of potato and pea ranges between 45 to 51%. The predicted amino acid sequence from each SSII cDNA contains the KXGGL consensus motif at the putative ADP-Glc binding site. Both clones also contain putative transit peptides followed by the VRAA(E)A motif, the consensus cleavage site located at the C-terminus of chloroplast transit peptides. The identity of the zSSIIa and zSSIIb clones as starch synthases was confirmed by expression of enzyme activity in Escherichia coli. Genomic DNA blot analysis revealed two copies of zSSIIa and a single copy of zSSIIb. zSSIIa was expressed predominantly in the endosperm, while transcripts for zSSIIb were detected mainly in the leaf at low abundance. These findings establish that the zSSIIa and zSSIIb genes are characteristically distinct from genes encoding granule-bound starch synthase I (Waxy protein) and starch synthase I.

Mercury-induced Conformational Changes and Identification of Conserved Surface Loops in Plasma Membrane Aquaporins from Higher Plants TOPOLOGY OF PMIP31 FROM BETA VULGARIS L.

Aquaporins are integral membrane proteins occurring in mammals, plants, and microorganisms, which serve as channels that permit the bidirectional passage of water through cellular membranes. Higher plants contain abundant levels of aquaporins in both the tonoplast and plasma membrane. Aquaporins contain six transmembrane segments with three surface loops located at the apoplastic face of the membrane and two loops at the cytosolic side. In this study, we probed the topology of plasma membrane aquaporins to determine the effects of divalent cations on aquaporin conformation, and to identify structural features that distinguish plasma membrane intrinsic proteins from tonoplast intrinsic proteins. Plasma membrane vesicles from storage tissue of Beta vulgaris L. were subjected to limited proteolysis, and proteolytic fragmentation patterns were detected using affinity-purified antibodies recognizing aquaporins of 31-kDa. In its native membrane-associated state, the 31-aquaporin band, PMIP31, was refractory to proteolysis by trypsin. However, mercuric compounds specifically induced a conformational change resulting in the exposure of a proteolytic cleavage site and formation of a unique 22-kDa proteolytic fragment (p22). N-terminal sequence analysis of p22 established its identity as an aquaporin-derived fragment. Topological studies using sealed right-side-out plasma membrane vesicles established that the proteolytic cleavage site is located at surface loop C, the second apoplastic loop, immediately preceding the sequence Gly-Gly-Gly-Ala-Asn. The Gly-Gly-Gly-Ala-Asn-X-X-X-X-Gly-Tyr motif of loop C and a 14 amino acid motif in apoplastic loop E, Thr-Gly-Ile/Thr-Asn-Pro-Ala-Arg-Ser-Leu/Phe-Gly-Ala-Ala-Ile/Val-Ile/Val-Phe/Tyr-Asn are completely conserved in all known higher plant aquaporins of plasma membrane origin and are not present in any of the known tonoplast intrinsic proteins. These results demonstrate that the two highly conserved plasma membrane intrinsic protein surface loops are structural features that clearly distinguish plasma membrane from tonoplast aquaporins.

Non-aqueous gel permeation chromatography of wheat starch in dimethylacetamide (DMAC) and LiCl: extrusion-induced fragmentation

Abstract Automated gel permeation chromatography (GPC) with application of the universal calibration concept was used to investigate the mechanism of extrusion-induced starch fragmentation in wheat. High and low protein flours were subjected to twin-screw extrusion and the effects of moisture, die temperature, screw speed, mass flow rate and protein content on starch structure and textural properties were investigated by non-aqueous GPC in dimethylacetamide (DMAC) and LiCl. This solvent system allowed for complete dissolution of the native and extruded starches. The use of refractive index and viscosity detectors enabled application of Mark-Houwink calculations to obtain quantitative size profiles of unprocessed and processed starch, and information describing branching patterns of the starch. Fragmentation was most pronounced in amylopectins of MW 10 7 –10 8 , which yielded fragments of MW 10 5 –10 7 . Of the operating parameters investigated, low die temperature and low moisture content led to extensive fragmentation. Methylation analysis showed only modest changes in linkage distributions, with little or no increase in terminal glucose indicating few fragmentation points relative to the total number of glycosidic linkages present. The lack of dextrins or oligosaccharides suggests that fragmentation occurs primarily in the B chains of amylopectin. Interactions between die temperature and moisture content were shown to significantly effect the hardness, cohesiveness, springiness, gumminess and chewiness of the extruded flours.

(1,3)-β-Glucan synthase fromSaccharomyces cerevisiae: In vitro activation byβ-lactoglobulin or Brij-35, and photoaffinity labeling of enriched microsomal fractions with 5-azido-UDP-Glc and 8-azido-GTP

Two new activators of (1,3)-β-glucan synthase fromSaccharomyces cerevisiae were identified, and a procedure for preparing enriched enzyme fractions by removal of peripheral membrane proteins and entrapped soluble proteins was developed. Microsomal enzyme activity, known to be enhanced by bovine serum albumin (BSA), was stimulated threefold by both β-lactoglobulin and Brij-35. Both apparently substituted for BSA, since no synergistic effects were observed with activators added in combination. Successive washings of microsomal fractions with the detergents Brij-35 and Tergitol NP-40 to remove peripheral and vesicle-entrapped proteins yielded particulate fractions five-fold enriched in glucan synthase activity. GTP, an important effector of glucan synthase, improved purification of the enzyme during detergent extractions. Various membrane fractions were photolabeled with 5[32P]N3UDP-Glc or 8N3[32P]GTP, and potential UDP-Glc and GTP-binding polypeptides were identified. However, further enrichment will be required to determine which of these might represent subunits of the yeast glucan synthase complex.

Characterization of the UDP-glucose: (1,3)-β-glucan (callose) synthase from plasma membranes of celery: polypeptide profiles and photolabeling patterns of enriched fractions suggest callose synthase complexes from various sources share a common structure

Abstract Plasma membranes were purified from celery ( Apium graveolens L.) by two-phase partitioning and the UDP-glucose (1,3)-β-glucan (callose) synthase was solubilized with the detergent CHAPS. The techniques of moderate pressure anion exchange chromatography (FPLC), gel filtration chromatography, affinity chromatography on UDP-agarose and product-entrapment were then investigated as means for further purification. Enriched fractions were obtained by anion exchange chromatography in 0.1% CHAPS and 50 mM Tris-HCl (pH 7.5, spec. act., 568 nmol min −1 (mg prot.) −1 ), by affinity chromatography using UDP-agarose (1405 nmol min −1 (mg prot.) −1 ) and by product entrapment (914 nmol min −1 (mg prot.) −1 ). Fractions purified by anion exchange chromatography contained numerous polypeptides, however callose synthase purified by the affinity step yielded a silver-stained profile of six prominent polypeptides of 26.8, 30.5, 35, 41, 56 and 72 kDa. In the product-entrapped sample, the polypeptides of 41, 56 and 72 kDa incorporated [ 32 P]5N 3 UDP-Glc in a Ca 2+ -dependent manner, implying that these could play a role in the binding of UDP-Glc to the enzyme. The polypeptides at 26.8, 30.5, 35 and 72 kDa have also been observed in purified preparations from various other sources and could represent other components of the callose synthase complex.

Peroxidative properties of betanin decolorization by cell walls of red beet

Abstract The effect of H 2 O 2 on betanin decolorization by horseradish peroxidase and a red beet cell wall preparation was examined. Both reactions were stimulated by the addition of micromolar concentrations of H 2 O 2 and were accompanied by the formation of an unstable product with an absorbance maximum of ca 452 nm. In the absence of added H 2 O 2 , the cell wall reaction was inhibited by catalase. When cell walls alone were placed in assay buffer, pH 3.4, H 2 O 2 generation occurred. The addition of betanin caused a further production of H 2 O 2 . These results suggest that the beet cell wall-mediated decolorization reaction proceeds via a peroxidatic mechanism.

Effect of glutaraldehyde on the activity of some DNA restriction endonucleases

The effect of the bifunctional crosslinking reagent glutaraldehyde on the activity of the restriction enzymes Bam HI,Hind III, EcoRI, and Tthlll I was investigated. The four enzymes exhibited differential sensitivity to inactivation. Tthlll I was the most sensitive, with activity losses occurring at levels of 0.0025% and above.Hind III was the most stable of the four and remained fully active at concentrations as high as 0.075%. Addition of BSA to incubation mixtures generally had a stabilizing effect. Implications of these results for the design of glutaraldehyde-based methods for the immobilization of restriction endonucleases are discussed.

Solubilization of a digitonin-stable glucan synthase from red beet root

Abstract UDP-glucose: 1,3-1,4 s-glucan synthase was solubilized from red beet (Beta vulgaris L.) root microsomes by treatment with 1% digitonin at 30°C. The enzyme shows unusual stability for a solubilized glucan synthase, with full activity retention after storage for 1 week at 4°C.

GENERATION AND APPLICATION OF IMMUNOLOGICAL PROBES FOR THE STUDY OF MEMBRANE-BOUND PROTEINS AND ENZYMES—A REVIEW

ABSTRACT Immunological techniques are widely used for the detection of enzymes in food systems and for elucidating protein structure and function. The generation of anti‐bodies recognizing membrane‐bound proteins is difficult due to their hydrophobic nature, and reduced antigenicity. This review summarizes strategies for the production and purification of polyclonal antibodies recognizing membrane proteins. Applications such as enzyme immunoprecipitation, use of site‐specific anti‐peptide. antibodies for determination of the topology of membrane‐embedded proteins, immunocytochemistry, and expression vector cloning are described.

Food science education in the USA: an academic perspective

Abstract In a multidisciplinary field such as food science, educators are faced with a unique set of challenges. Incoming students at both the undergraduate and graduate levels come from diverse backgrounds, with widely varying knowledge of ‘the basics’ of food science, and thus widely differing needs. Furthermore, there is a need to educate the public to improve the image and understanding of food science, in order to attract students to the field. This Viewpoint considers the problems faced by both students and educators in ensuring that graduates will have the skills to compete in todays marketplace.