A purified 75-kDa myrosinase and a crude rapeseed myrosinase fraction were used as antigens to produce mouse anti-myrosinase monoclonal antibodies. The 75-kDa myrosinase was also used to produce a polyclonal rabbit antiserum. The antiserum and one monoclonal antibody reacted with three distinct rapeseed polypeptides of 75,70 and 65 kDa (M75, M70 and M65, respectively). A second set of monoclonal antibodies reacted exclusively with the 75-kDa form of myrosinase, and a third set showed specificity towards two components of 52 and 50 kDa (myrosinase-binding proteins, MBP52 and MBPSO, respectively). MBP52 and MBP5O lack inherent myrosinase activity, but are nevertheless capable of mediating immunoprecipitation of myrosinase due to their interaction with myrosinase. Gel chromatography and glycerol gradient centrifugation experiments resolved two myrosinasecontaining fractions. One of these had an approximate molecular mass of 140 kDa and consisted of disulfidelinked dimers of the 75-kDa myrosinase. The other fraction was heterogeneous in size with molecular masses ranging from 250 kDa to approximately 1 MDa. The high-molecular-mass fractions contained complexes consisting of disulfide-linked 70-kDa and 65-kDa myrosinases and non-covalently bound 52-kDa and 50-kDa myrosinase-binding proteins.Many cruciferous plants contain glucosinolates. These are low-molecular-mass compounds consisting of a glucose residue linked via a thioglucoside bond to an amino acid derivative. Some 80 different glucosinolates have been identified, differing mainly in their amino acid portions [l]. The glucosinolates seem to always be accompanied by a group of isoenzymes termed thioglucoside glucohydrolase (EC 3.2.3.1) but mostly referred to by their trivial name myrosinase. This enzyme is capable of catalyzing the hydrolysis of glucosinolates to form glucose, sulfate and, for example, thiocyanates, isothiocyanates, nitriles or epithionitriles. The exact outcome of the reaction is dependent on the substrate as well as on the reaction conditions used. Several of the products have potentially goitrogenic and hepatotoxic effects, which severely restrict the use of rapeseed protein concentrates as animal fodder. Myrosinase has previously been purified from rapeseed and found to be a glycoprotein with a molecular mass of 135 kDa, consisting of two 65-kDa polypeptide chains and containing about 14% carbohydrate [2]. There is evidence for glucosinolates and their degradation products beeing important in determining the specificity of interaction between crucifers and their potential herbivores, pathogens, competitors and symbionts [3]. To understand the physiological significance of the glucosinolate/myrosinase system it is important to study both components. We have produced Abbreviations NaC1/Pi, phosphate-buffered saline, i.e. 8 mM Na2HP04, 1.5 mM KH2P04, 3 mM KC1, 140 mM NaC1, pH 6.8; Tween 20, polyoxyethylene sorbitan monolaurate; ESP, epithiospecifier protein; M75, M70, M65, myrosinases with subunit molecular masses of 75, 70 and 65-kDa, respect...

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5' control region of the napA gene were fused to the reporter gene beta-glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between -1101 and -309 resulted in increased GUS activity. In contrast, deletion of sequences between -309 and -211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between -152 and +44. Further 5' deletion of the fragment to -126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between -148 to -120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to -152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.

We have cloned and characterized a cDNA from Arabidopsis thaliana that most likely encodes a novel member of the vast superfamily of G-protein-coupled receptor proteins (GPCRs). By taking advantage of amino acid sequence similarities between plant expressed sequence tags (ESTs) and established G-proteincoupled receptor sequences, a probe was obtained which was used for the screening of an Arabidopsis cDNA library. The cDNA which was found is very infrequently represented in the cDNA library, suggesting a low and/or spatially restricted expression. A region of the translated sequence of the cDNA shows the highest similarity to CAMP receptors from the slime mold Dictyostelium discoideurn. The same region is also similar to that in members of the animal calcitonin family of receptors. Another region of the putative receptor, however, is similar to sequences of serotonin receptors and other receptors of the socalled rhodopsin family of GPCRs. The rhodopsin family has numerous members in higher vertebrate species. Alignments and phylogenetic analyses of the regions of similarity yielded results in accordance with other evolutionary considerations. Our cDNA thus occurred on a distinct major branch in relation to the rest of the rhodopsin family. In relation to the calcitonin family, our cDNA and CAMP receptors occurred together on a distinct major branch but appear to have diverged from each other shortly after their divergence from the rest of the calcitonin family. Other features further argue for a tentative identification of it as a GPCR. It displays seven discrete and strongly predicted transmembrane domains when analyzed in hydropathy plots. The preferred orientation is with the amino terminus towards the outside. It has one Cys residue in extracellular loop 1 and another in extracellular loop 2. Cys residues in these loops are known to form disulfide bridges in many other GPCRs. Finally, it has several fully conserved amino acids that belong to the most conserved in previously known GPCRs, that occur in the above regions of similarity.

No abstract

A study by crossed immunoelectrophoresis performed in conjunction with precipitate excision and polypeptide analysis identified a new antigen complex in the envelope of Escherichia coli ML308-225. This antigen corresponds to antigen 43 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978). Immunoprecipitation experiments conducted with specific antiserum revealed that the complex was expressed on the cell surface and that it contained, in equal stoichiometry, two chemically distinct polypeptides termed a and (Mrs of 60,000 and 53,000, respectively). The polypeptide was heat modifiable, displaying an apparent Mr of 37,000 when solubilized at temperatures below 70°C. Analysis of fractions obtained following cell disruption, isopycnic centrifugation, and detergent extraction indicated that both a and polypeptides were components of the outer membrane. The two polypeptides were not linked by disulfide bonds, and neither was peptidoglycan associated. The complex contained no detectable lipopolysaccharide, enzyme activity, fatty acyl groups, or other cofactors. Neither correlated with E. coli proteins of similar molecular weight which had previously been shown to be associated with the outer membrane. Antibodies were raised to individual a and I1 polypeptides. Each of these sera was shown to be subunit specific when tested against denatured membrane proteins. In contrast, each immunoglobulin preparation coprecipitated both a and polypeptides when tested against undenatured proteins derived from Triton X-100-treated membranes. The results reveal the presence of a novel bipartite protein antigen in the outer membrane of E. coli.The outer membrane of Escherichia coli is known to contain a number of major protein species (1, 15). Several of these are porins involved in the passive accumulation of small hydrophilic solutes (2,19). Others, such as the ironregulated outer membrane proteins and the vitamin B12 receptor, are involved in the TonB-dependent uptake of more specific solutes (4). Other major proteins of the E. coli outer membrane include the Braun lipoprotein (the Lpp protein), peptidoglycan-associated lipoprotein, and the ompA gene product. In part at least, these appear to play a structural role for the cell envelope (1, 15). Several other minor proteins and enzymes have also been identified and studied (10,17,20,35). In addition to the above components, there exist in the outer membratne several additional proteins about which considerably less is known, e.g., protein III, an 83-kilodalton iron-regulated protein, and several proteins in the 60-to 40-kilodalton range (15,26).In an attempt to identify and characterize the major antigens of the cell envelope of aerobically grown E. coli, a crossed immunoelectrophoresis (CIE) reference profile has been established in which over half of the 50 or so resolved antigens have been identified in functional terms or partially characterized (for reviews, see references 21 to 23).