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61.
Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of Mr=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (Mr=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP
dithiobis(succinimidyl propionate)
- EDTA
ethylenediaminetetraacetic acid
- ER
endoplasmic reticulum
- kDa
kilodalton
- Mr
relative molecular mass
- PHA
phytohemagglutinin
- SDS
sodium dodecylsulfate
- PAGE
polyacrylamide gel electrophoresis 相似文献
62.
Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB
antiserum to integral protein-body membrane proteins
- anti-G
antiserum to integral glyoxysomal membrane proteins
- anti-L
antiserum to alkaline lipase
- ER
endoplasmic reticulum
- Mr
relative molecular mass
- mRNA
poly(A)-rich messenger RNA
- PAGE
polyacrylamide gel electrophoresis
- poly(A)
polyadenylic acid
- SDS
sodium dodecyl sulphate 相似文献
63.
When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the
total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial
characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to
homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins.
Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular
masses (Mrs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous
on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products)
in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins
appear to be produced late in seed development but are capable of being induced early in development by culturing embryos
in vitro and are markedly enhanced by ABA. 相似文献
64.
The kinetics of inhibition by protein- and RNA-synthesis inhibitors (cycloheximide and cordycepin, respectively) of indole-3-acetic acid (IAA)-induced elongation growth were investigated using abraded coleoptile segments of Zea mays L. Removal of the cuticle — a diffusion barrier for solutes — by mechanical abrasion of the outer epidermal cell wall increased the effectiveness of inhibitors tremendously. In an attempt to elucidate the role of growth-limiting protein(s) (GLP) in the growth mechanism the following results were obtained. The elongation induced by IAA was completely inhibited when cycloheximide (10 mol·l-1) was applied to abraded coleoptile segments as shortly as 10 min before the onset of the growth response (=5 min after administration of IAA). However, when cycloheximide was applied after 60 min of IAA treatment (when a steady-state growth rate is reached), the time required for complete cessation of growth was much longer (about 40 min). Cycloheximide inhibited the incorporation of [3H]leucine into protein within about 5 min. Cordycepin (400 mol·l-1) prevented IAA-induced growth when applied as shortly as 25 min before the onset of the growth response (=10 min before administration of IAA) but required more than 60 min for a full inhibition of steady-state growth. The incorporation of [3H]adenosine into RNA was inhibited by cordycepin within 10 min. It is concluded that, contrary to previous investigations with nonabraded organ segments, the initiation of growth by IAA depends directly on the synthesis of GLP. Moreover, the apparent lifetime of GLP is at least four times longer than the time required by cycloheximide to inhibit the initiation of growth by IAA. This is interpreted to mean that GLP is not present before IAA starts to act but is synthesized as a consequence of IAA action starting a few minutes before the initiation of growth. Interpreting the kinetics of growth inhibition by cordycepin in a similar way, we further conclude that GLP synthesis is mediated by IAA-induced synthesis of the corresponding mRNA which starts about 10 min before the onset of GLP synthesis. Inhibition by cycloheximide and cordycepin of IAA-induced growth cannot be alleviated by acidifying the cell wall to pH 4-5, indicating that these inhibitors do not act on growth via an inhibition of auxin-mediated proton excretion.Abbreviations CHI
cycloheximide
- COR
cordycepin
- GLP
growth-dimiting protein(s)
- IAA
indole-3-acetic acid
- mRNAGLP
mRNA coding for GLP 相似文献
65.
The glue protein of ribbed mussels (Geukensia demissa): a natural adhesive with some features of collagen 总被引:1,自引:0,他引:1
J. Herbert Waite Douglas C. Hansen Kathleen T. Little 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1989,159(5):517-525
Summary The Atlantic ribbed musselGeukensia (Modiolus)demissa attaches itself to the roots of cord grass and other hard objects in tidal salt marshes by spinning adhesive byssal threads. The precursor of a protein apparently present in the adhesive plaques of the threads was isolated in quantity from the foot of the mussel. The protein has an apparent molecular weight of 130000, a pI of 8.1, and contains a high proportion of Gly, Glu/Gln, Lys and 3,4-dihydroxyphenyl-l-alanine (DOPA). Sequence of tryptic peptides suggests a pattern of repeated motifs, such as: Gly-DOPA-Lys, and X-Gly-DOPA-Y-Z-Gly-DOPA/Tyr-Lys, where X is Thr or Ala in octapeptides and Gln-Thr in nonapeptides. Y is variable, but more often than not hydrophobic; and Z is frequently Pro or 4-trans-hydroxyproline (Hyp). The presence of Pro-Gly and Hyp-Gly sequences of -hydroxylysine in the protein is reminiscent of typical collagens; however, the protein is not labile to clostridial collagenase, nor does collagen cross-react with antibodies raised against the mussel protein. Unlike typical collagens, Gly probably occurs only at every 4th or 5th residue in this unusual mussel protein.Abbreviations
Anti-Gdfp
anti-G. demissa foot protein
-
Dopa
3,4-dihydroxyphenylalanine
-
CTAB
cetyltrimethylammonium bromide
-
HPLC
high performance liquid chromatography
-
PAGE
polyacrylamide gel electrophoresis
Supported by grants from the National Science Foundation (DMB 8500301) and the Office of Naval Research (N00014-86K-0717) 相似文献
66.
A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae 总被引:4,自引:0,他引:4
Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10%
(exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products
by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases
exhibited identical mobility, each one consisting of two polypeptides with M
r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised
against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products
with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases
with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts.
These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same
gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond
the permeability barrier of the cell. 相似文献
67.
Helmut Krüger Werner Schr?der Klaus Buchner Ferdinand Hucho 《Journal of Protein Chemistry》1990,9(4):467-473
Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC50 of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser38-Arg74 and His107-Lys148. Each of the inhibiting fragments contains an intact Ca2+-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist. 相似文献
68.
69.
Aurelio Serrano Patricia Giménez Siegfried Scherer Peter Böger 《Archives of microbiology》1990,153(6):614-618
The in situ location of the electron carrier protein cytochrome C
553 (cyt c
553) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c
553 specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c
553 during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c
553 is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c
553
cytochrome c
553
- PBS
phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4)
- PMSF
phenylmethylsulfonyl fluoride
Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz. 相似文献
70.
Protein phosphorylation in Streptomyces albus 总被引:1,自引:0,他引:1
Z. Dobrová M. Jirsová T. Petík P. Ryavý J. Náprstek J. Janeek 《FEMS microbiology letters》1990,71(1-2):145-148
The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein. 相似文献