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51.
52.
The location of the (13)--glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (13)--glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (13, 14)--glucan-BSA conjugate. Binding was inhibited by (13)--oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (13)--linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 104M–1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (13)--glucan in the inner wall layer of thin sections of the N. alata pollen tubes.Abbreviations BSA
bovine serum albumin
- PBS
phosphate-buffered saline
- ELISA
enzyme linked immunosorbent assay
- DP
degree of polymerization
- PVC
polyvinyl chloride
P.J.M. is an Australian Postdoctoral Research Fellow. We wish to thank Joan Hoogenraad for her technical assistance with the tissue culture, and Althea Wright for her assistance in the preparation of this paper. 相似文献
53.
P J Meikle K F Ng E Johnson N J Hoogenraad B A Stone 《The Journal of biological chemistry》1991,266(33):22569-22581
The membrane-bound beta-glucan synthase from Italian ryegrass (Lolium multiflorum L.) endosperm cells has been solubilized by both non-ionic and zwitterionic detergents. A complex relationship exists between the ratio of (1----3)-, (1----4)-, and (1----3, 1----4)-beta-glucan products of the solubilized enzyme, the cations present, and the concentration of the uridine 5'-diphosphoglucose substrate. Monoclonal antibodies directed against the beta-glucan synthase complex were generated by immunization of mice with an unfractionated microsomal reparation. Hybridoma cell lines were screened using a combination of indirect enzyme-linked immunosorbent assay followed by an enzyme-capture assay. The purified monoclonal antibodies were used with Pan-sorbin (stablized protein A-bearing staphylococcal cells) to immunoprecipitate an active beta-glucan synthase complex which had been solubilized from a microsomal preparation with 0.6% CHAPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated synthase complex revealed four major polypeptides of apparent molecular mass 30, 31, 54, and 58 kDa together with several minor components. The immunoprecipitated beta-glucan synthase complex was capable of synthesizing both (1----3)- and (1----4)-beta-glucans. A new photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose, 5-[3-(p-azidosalicylamide]allyl-uridine 5'-diphosphoglucose was synthesized in a three-step reaction sequence involving mercuration of UDP-Glc, alkylation of 5-Hg-UDP-Glc, and acylation of 5-(3-amino)allyl-UDP-Glc and characterized by chemical and spectroscopic analysis. The analogue inhibits (Kiapp 16 microM) and, upon UV irradiation, irreversibly inactivates the beta-glucan synthase. The analogue was iodinated with Na125I to give a radiolabeled, photoreactive compound, and was used in photoaffinity labeling of UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and several putative UDP-Glc-binding proteins from L. multiforum. The radiolabeled analogue specifically labeled the 31-kDa polypeptide in the immunoprecipitated synthase complex. The photolabeling of this polypeptide is strictly dependent on UV irradiation, is blocked by uridine 5'-diphosphoglucose and uridine 5'-diphosphate, and reaches saturation at analogue concentrations above 300 microM. These results indicate that the 31-kDa polypeptide in the beta-glucan synthase complex bears a uridine 5'-diphosphoglucose-binding site and is involved in the catalysis of beta-glucan synthesis. 相似文献
54.
Analysis of seventeen yeast strains by 13C-NMR spectroscopy has confirmed the significance of glycerol as the sole osmoregulatory solute under salt-stressed conditions, and has shown arabitol to be present in most of the osmotolerant species. Ribitol was detected in some species, including Debaryomyces hansenii, although ribitol accumulation did not correlate with the osmotic pressure of the medium. Relative amounts of arabitol and ribitol decreased in relation to glycerol when the external osmotic pressure was increased. Trehalose was present during exponential growth of some species. 相似文献
55.
Summary Incubation period in mice of ME7 scrapie agent is known to be controlled by the gene sinc. Evidence is presented that the same gene probably also determines incubation period for the 22A scrapie agent which has very different biological properties. Not only is there a reversal of the direction of gene effect when these agents are compared in mice homozygous for two different alleles of sinc, but there is also a different type of allelic interaction—absence of dominance with ME7 and overdominance with 22A. Other agents are mentioned which extend this range of allelic interaction even further. It is suggested that replication of scrapie agent depends on a multimeric host site and that the two alleles of sinc contribute different types of subunits to these sites. 相似文献
56.
57.
Background
Amino acid repeat-containing proteins have a broad range of functions and their identification is of relevance to many experimental biologists. In human-infective protozoan parasites (such as the Kinetoplastid and Plasmodium species), they are implicated in immune evasion and have been shown to influence virulence and pathogenicity. RepSeq is a new database of amino acid repeat-containing proteins found in lower eukaryotic pathogens. The RepSeq database is accessed via a web-based application which also provides links to related online tools and databases for further analyses. 相似文献58.
Kakavanos R Lehn P Callebaut I Meikle PJ Parkinson-Lawrence EJ Hopwood JJ Brooks DA 《FEBS letters》2006,580(1):87-92
Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites. 相似文献
59.
Michelle S Teng Martijn PJ Dekkers Ling Bee Ng Suzanne Rademakers Gert Jansen Andrew G Fraser John McCafferty 《BMC biology》2006,4(1):22-9
Background
G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro systems. Caenorhabditis elegans is a soil-dwelling, bacteria-feeding nematode that uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds, making this an ideal system for studying in vivo GPCR-ligand interactions. We sought to test this by functionally expressing two medically important mammalian GPCRs, somatostatin receptor 2 (Sstr2) and chemokine receptor 5 (CCR5) in the gustatory neurons of C. elegans. 相似文献60.
PJ Waller B-L Ljungström O Schwan L Rudby Martin DA Morrison A Rydzik 《Acta veterinaria Scandinavica》2006,47(1):23-10