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Purification and characterization of Dolichos lablab lectin 总被引:1,自引:0,他引:1
The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has
been purified from seeds of Dolichos lablab (hyacinth bean) to
electrophoretic homogeneity by affinity chromatography on an ovalbumin-
Sepharose 4B column. The purified lectin gave a single symmetric protein
peak with an apparent molecular mass of 67 kDa on gel filtration
chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE.
N-Terminal sequence analysis of these bands revealed subunit heterogeneity
due to posttranslational proteolytic truncation at different sites mostly
at the carboxyl terminus. The carbohydrate binding properties of the
purified lectin were investigated by three different approaches:
hemagglutination inhibition assay, quantitative precipitation inhibition
assay, and ELISA. On the basis of these studies, it is concluded that the
Dolichos lablab lectin has neither an extended carbohydrate combining site,
nor a hydrophobic binding site adjacent to it. The carbohydrate combining
site of DLL appears to most effectively accommodate a nonreducing terminal
alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6
equatorial hydroxyl groups of alpha-d-mannopyranosyl and
alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but
not IgG, and the recent finding that this lectin interacts specifically
with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor
and preserves human cord blood stem cells and progenitors in a quiescent
state for prolonged periods in culture, make this lectin a valuable tool in
biomedical research.
相似文献
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We have broadly defined the DNA regions regulating esterase6 activity in
several life stages and tissue types of D. melanogaster using P-
element-mediated transformation of constructs that contain the esterase6
coding region and deletions or substitutions in 5' or 3' flanking DNA.
Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and
the primary sequences regulating its activity lie between -171 and -25 bp
relative to the translation initiation site: deletion of these sequences
decrease activity approximately 20-fold. Hemolymph activity is also
modulated by four other DNA regions, three of which lie 5' and one of which
lies 3' of the coding region. Of these, two have positive and two have
negative effects, each of approximately twofold. Esterase6 activity is
present also in two male reproductive tract tissues; the ejaculatory bulb,
which is another ancestral activity site, and the ejaculatory duct, which
is a recently acquired site within the melanogaster species subgroup.
Activities in these tissues are at least in part independently regulated:
activity in the ejaculatory bulb is conferred by sequences between -273 and
-172 bp (threefold decrease when deleted), while activity in the
ejaculatory duct is conferred by more distal sequences between -844 and
-614 bp (fourfold decrease when deleted). The reproductive tract activity
is further modulated by two additional DNA regions, one in 5' DNA (-613 to
-284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to
+2731 bp; threefold decrease when deleted) that probably overlaps the
adjacent esteraseP gene. Collating these data with previous studies
suggests that expression of EST6 in the ancestral sites is mainly regulated
by conserved proximal sequences while more variable distal sequences
regulate expression in the acquired ejaculatory duct site.
相似文献
6.
Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans 总被引:2,自引:0,他引:2
Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and
D. simulans for two common allozyme forms, as well as for several other
less common variants. Parallel latitudinal clines in the frequencies of the
common EST6-F and EST6-S allozymes in these species have previously been
interpreted in terms of a shared amino acid polymorphism that distinguishes
the two variants and is subject to selection. Here we compare the sequences
of four D. simulans Est-6 isolates and show that overall estimates of
nucleotide heterozygosity in both coding and 5' flanking regions are more
than threefold higher than those obtained previously for this gene in D.
melanogaster. Nevertheless, the ratio of replacement to exon silent-site
polymorphism in D. simulans is less than the ratio of replacement to silent
divergence between D. simulans and D. melanogaster, which could be the
result of increased efficiency of selection against replacement
polymorphisms in D. simulans or to divergent selection between the two
species. We also find that the amino acid polymorphisms separating EST6- F
and EST6-S in D. simulans are not the same as those that separate these
allozymes in D. melanogaster, implying that the shared clines do not
reflect shared molecular targets for selection. All comparisons within and
between the two species reveal a remarkable paucity of variation in a
stretch of nearly 400 bp immediately 5' of the gene, indicative of strong
selective constraint to retain essential aspects of Est-6 promoter
function.
相似文献
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The activity of the soluble enzyme CPK and the membrane bound enzyme Na+-K+ ATPase as a function of storage temperature, time of storage and cryoprotectant type and concentration in canine myocardial tissue was invesigated. Activity of CPK is well preserved at ?196 °C and ?79 °C and falls off during one month storage at ?40 °, ?20 °, and 0 °C. Na+-K+ ATPase demonstrates a greater liability. After an initial cryoprotectant “activation,” activity drops. In all cases, however, addition of the cryoprotectant preserved activity better than in samples stored only in buffer. 相似文献
9.
The lower lethal temperature of many insects indicates an overwintering flexibility as a result of either extensive supercooling or production of cryoprotectants. Ontogenetically, the gall fly (Eurosta solidagensis) utilizes both means of seasonal cryoprotection. All stages except third instar larvae demonstrate supercooling points well below the lowest temperature normally experienced by that particular stage. The third instar larvae exhibit a high supercooling point but are well protected by a cryoprotectant system consisting of glycerol, sorbitol, and trehalose. Glycerol is accumulated, possibly from triglyceride sources, during early autumn and reaches plateau levels (0·6 M) by early winter. Sorbitol synthesis is delayed until freezing exposures and reaches a plateau with glycerol at 0·3 M. It is not until mid-winter that peak trehalose levels are reached (300 mg %). All cryoprotectant levels are a reflection of haemolymph concentrations.Laboratory acclimation experiments further quantify these results. Trehalose synthesis is time and temperature dependent and appears to be affected by developmental processes. 相似文献
10.
The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity. 相似文献