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...
Human growth hormone binds two receptor molecules and thereby induces signal transduction through receptor dimerization. At high concentrations, growth hormone acts as an antagonist because of a large difference in affinities at the respective binding sites. This antagonist action can be enhanced further by reducing binding in the low affinity binding site. A growth hormone antagonist mutant Gly-120 3 Arg, has been crystallized with its receptor as a 1:1 complex and the crystal structure determined at 2.9 Å resolution. The 1:1 complex is remarkably similar to the native growth hormone-receptor 1:2 complex. A comparison between the two structures reveals only minimal differences in the conformations of the hormone or its receptor in the two complexes, including the angle between the two immunoglobulin-like domains of the receptor. Further, two symmetry-related 1:1 complexes in the crystal form a 2:2 complex with a large solvent inaccessible area between two receptor molecules. In addition, we present here a native human growth hormone-human growth hormone-binding protein 1:2 complex structure at 2.5 Å resolution. One important difference between our structure and the previously published crystal structure at 2.8 Å is revealed. Trp-104 in the receptor, a key residue in the hormone-receptor interaction, has an altered conformation in the low affinity site enabling a favorable hydrogen bond to be formed with Asp-116 of the hormone.
The distribution of napin and crucifenn, the two major storage proteins in rape seed, Brassica napus, has been visualized during seed development by antibody staining of paraffin-embedded and sectioned seeds. The results indicate that the synthesis of both proteins during embryogenesis is strictly regulated with respect to time and tissue. Although the synthesis of napin started a few days earlier than that of cruciferin, both proteins displayed similar pattems in their spatial distributions. They were first detected in the axis, then in the outer cotyledon, and finally in the cells of the inner cotyledon. Both proteins are also present in the endosperm, although in lower amounts. In germinating seeds, napin and crucifenn were rapidly degraded. Within 2 days the amounts had decreased dramatically, and after 4 days hardly any cells contained napin or cruciferin. Biochemical analyses of dissected embryos showed that, for napin as well as for cruciferin, similar levels of polypeptides were found in the axis and cotyledons.The two major types of storage proteins in rape seed (Brassica napus) are the 2S albumin, napin, and the 12S globulin, cruciferin (4,8,13,17
In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes i n embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75,73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that i n 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70-and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66-and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only i n developing seeds, whereas MB genes were most highly expressed i n seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. hybridization analysis of B. napus embryos showed that MA transcripts were present predominantly i n myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axis contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70-and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of their transcription should provide interesting information concerning a complex type of gene regulation.Myrosinases, or thioglucoside glucohydrolases (EC 3.2.3. I), are isoenzymes that catalyze the hydrolysis of glucosinolates, a group of low molecular mass compounds consisting of a Glc residue linked to a variety of side chains, which are mainly amino acid derivatives. Upon hydrolysis of glucosinolates by the enzyme myrosinase, Glc, sulfate, and either '
In the solanaceous plant Nicotiana alata, self-incompatibility is controlled by a single, multiallelic locus (S locus) expressed in both pollen and pistil. Previously, we have shown cosegregation between alleles of the S locus and alleles of a gene that encodes a glycoprotein with ribonuclease activity (S-RNase). Furthermore, expression of the S-RNase gene is apparently confined to the pistil and is correlated with the onset of self-incompatibility. In this paper, we report that the S-RNase gene is also expressed at low levels in developing pollen. A transcript in developing pollen hybridized to a cDNA encoding the SrRNase allele of the parent plant and did not hybridize to cDNAs encoding other S-RNase alleles. Two cDNAs for the S2-RNase were cloned from a library derived from anthers of a plant homorygous for the S2 allele and both corresponded to the coding sequence of the SrRNase. The product of the S-RNase gene was detected by immunocytochemistry in the intine of mature, hydrated pollen grains. These results are interpreted in the light of current knowledge of the structure of the S locus.
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