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21.
Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties. 总被引:4,自引:3,他引:1 下载免费PDF全文
Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor. 相似文献
22.
Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans 总被引:1,自引:0,他引:1
The egg jelly coats of sea urchins contain sulfated fucans which bind to a
sperm surface receptor glycoprotein to initiate the signal transduction
events resulting in the sperm acrosome reaction. The acrosome reaction is
an ion channel regulated exocytosis which is an obligatory event for sperm
binding to, and fusion with, the egg. Approximately 90% of individual
females of the sea urchin Strongylocentrotus purpuratus spawned eggs having
only one of two possible sulfated fucan electrophoretic isotypes, a slow
migrating (sulfated fucan I), or a fast migrating (sulfated fucan II)
isotype. The remaining 10% of females spawned eggs having both sulfated
fucan isotypes. The two sulfated fucan isotypes were purified from egg
jelly coats and their structures determined by NMR spectroscopy and
methylation analysis. Both sulfated fucans are linear polysaccharides
composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I
is entirely sulfated at the O -2 position but with a heterogeneous
sulfation pattern at O -4 position. Sulfated fucan II is composed of a
regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p -
2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-
1]n. Both purified sulfated fucans have approximately equal potency in
inducing the sperm acrosome reaction. The significance of two structurally
different sulfated fucans in the egg jelly coat of this species could
relate to the finding that the sperm receptor protein which binds sulfated
fucan contains two carbohydrate recognition modules of the C-type lectin
variety which differ by 50% in their primary structure.
相似文献
23.
Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications 总被引:1,自引:0,他引:1
Evidence of associations between free-living amoebas and human disease has
been increasing in recent years. Knowledge about phylogenetic relationships
that may be important for the understanding of pathogenicity in the genera
involved is very limited at present. Consequently, we have begun to study
these relationships and report here on the phylogeny of Hartmannella
vermiformis, a free-living amoeba that can harbor the etiologic agent of
Legionnaires' disease. Our analysis is based on studies of small-subunit
ribosomal RNA genes (srDNA). Nucleotide sequences were determined for
nuclear srDNA from three strains of H. vermiformis isolated from the United
Kingdom, Germany, and the United States. These sequences then were compared
with a sequence previously obtained for a North American isolate by J. H.
Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an
average GC content of 49.6%. Sequence differences among the strains range
are 0.38%-0.76%. Variation occurs at 19 positions and includes 2
single-base indels plus 14 monotypic and 3 ditypic single-base
substitutions. Variation is limited to eight helix/loop structures
according to a current model for srRNA secondary structure. Parsimony,
distance, and bootstrap analyses used to examine phylogenetic relationships
between the srDNA sequences of H. vermiformis and other eukaryotes
indicated that Hartmannella sequences were most closely related to those of
Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent
with a separation between European and North American strains of
Hartmannella, but results of other tests of this relationship were
statistically inconclusive.
相似文献
24.
Studies on the binding of iron by rabbit intestinal microvillus membranes. 总被引:1,自引:0,他引:1 下载免费PDF全文
1. 59Fe binding by microvillus membranes purified from rabbit intestine was studied by means of a microfiltration procedure. 2. Binding activity from ferrous ascorbate chelates was 100-fold greater than from ferric chelates of citrate and nitrilotriacetate. Dual-label experiments indicated dissociation of iron complexes before binding to the membranes. 3. Binding was inhibited at low incubation temperatures and was optimal at neutral pH. 4. Binding activity was reduced in ileal preparations when compared with membranes prepared from proximal intestine. 5. Initial binding velocity followed saturation kinetics over the range 45-450 microM-iron: it was weakly inhibited in the presence of excess Co2+ and V3+. 6. The data provide additional evidence for high-affinity iron-binding sites on the intestinal microvillus membrane and indicate properties that may reflect the functional significance of the binding step in the absorption pathway for iron. 相似文献
25.
Rabbit intestinal microvillus membranes possess high-affinity receptors for iron whose activity reflects homeostatic changes in mucosal iron transport. To isolate and characterize these membrane components, purified microvilli were radiolabelled with 59Fe(II) and solubilized in Triton X-100. 59Fe in 105000g supernatants co-eluted with a major broad protein peak (Mr approx. 100000) on gel-permeation chromatography and was rendered diffusible by Pronase digestion but not mild periodate degradation. Fluorescence studies with castor-bean lectin conjugates showed specific binding of this affinity probe exclusively to brush-border membranes in the intestinal epithelium. Affinity chromatography of solubilized membrane proteins showed binding to columns of immobilized lectin. Elution with D(+)-galactose released glycoprotein-bound 59Fe purified up to sevenfold over initial membrane extracts. The lectin bound up to 82% of protein-bound 59Fe. In contrast polyspecific antisera raised against rabbit microvilli in guinea-pigs precipitated less than 10% of solubilized radioactivity. Significantly more protein-bound 59Fe in detergent extracts of microvilli purified from bled animals interacted specifically with the lectin, suggesting that membrane glycoprotein receptors are involved in the homeostatic control of intestinal iron transport. 相似文献
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28.
TM?Matthews RK?Duncan M?Zidanic TH?Michael PA?FuchsEmail author 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》2005,191(6):491-503
In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC50=73.3±5.0 pM) and d-tubocurarine (IC50=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates. 相似文献
29.
Abandoning ineffective medical practices and mitigating the risks of untested practices are important for improving patient health and containing healthcare costs. Historically, this process has relied on the evidence base, societal values, cultural tensions, and political sway, but not necessarily in that order. We propose a conceptual framework to guide and prioritize this process, shifting emphasis toward the principles of evidence-based medicine, acknowledging that evidence may still be misinterpreted or distorted by recalcitrant proponents of entrenched practices and other biases. 相似文献
30.
Elisabeth APM Romme David A McAllister John T Murchison Edwin JR Van Beek George S Petrides Cameron OS Price Erica PA Rutten Frank WJM Smeenk Emiel FM Wouters William MacNee 《Respiratory research》2013,14(1):129