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1.
Summary The preceding paper [30] shows that transepithelial ileal SO4 transport involves Na-dependent uptake across the ileal brush border, and Cl-dependent efflux across the serosal border. The present study examines more closely the serosal efflux process. Transepithelial mucosa (m)-to-serosa (s) ands-to-m fluxes (J
ms,J
sm) across rabbit ileal mucosa were determined under short-circuit conditions. SO4 was present at 0.22mm. In standard Cl, HCO3 Ringer's,J
ms
SO4
was 81.3±5.3 (1se) andJ
ms
SO4
was 2.5±0.2 nmol cm–2 hr–1 (n=20). Serosal addition of 4-acetamido-4-isothiocyanostilbene-22-disulfonate (SITS), 44-diisothiocyanostilbene-22-disulfonate (DIDS) or 1-anilino-8-naphthalene-sulfonate (ANS) inhibited SO4 transport, SITS being the most potent. Several other inhibitors of anion exchange in erythrocytes and other cells had no effect on ileal SO4 fluxes. In contrast to its effect on SO4 transport, SITS (500 m) did not detectably alter Cl transport.Replacement of all Cl, HCO3 and PO4 with gluconate reducedJ
ms
SO4
by 70% and increasedJ
ms
SO4
by 400%. A small but significantJ
net
SO4
remained.J
ms
SO4
could be increased by addition to the serosal side of Cl, Br, I, NO3 or SO4. The stimulatory effect of all these anions was saturable and SITS-inhibitable. The maximalJ
ms
SO4
in the presence of Cl was considerably higher than in the presence of SO4 (73.1 and 42.2 nmol. cm–2 hr–1, respectively;p<0.001). TheK
1/2 value for Cl was 7.4mm, 10-fold higher than that for SO4 (0.7mm). Omitting HCO3 and PO4 had no measurable effects on SO4 fluxes.This study shows that (i) SO4 crosses the serosal border of rabbit ileal mucosa by anion exchange; (ii) the exchange process is inhibited by SITS, DIDS and ANS, but not by several other inhibitors of anion exchange in other systems; (iii) SO4 may exchange for Cl, Br, I, NO3 and SO4 itself, but probably not for HCO3 or PO4; (iv) kinetics of the exchange system suggest there is a greater affinity for SO4 than for Cl, although the maximal rate of exchange is higher in the presence of Cl; and, finally (v) SITS has little or no effect on net Cl transport. 相似文献
2.
Gregory S. Harper Tina Rozaklis Julie Bielicki John J. Hopwood 《Glycoconjugate journal》1993,10(5):407-415
Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzymeN-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na
2
35
SO4. The addition of recombinantN-acetylgalactosamine-4-sulfatase to the media of35S labelled fibroblasts degraded up to 82% of the stored dermatan [35S] sulfate over a subsequent 96 h chase and released inorganic [35S] sulfate into the medium. In the presence of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [35S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na2SO4 and Na2MoO4. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzymein vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.Abbreviations SITS
4-acetamido-4-isothiocyanatostilbene-2,-2-disulfonic acid
- GAG
glycosaminoglycan
- 4S
N-acetylgalactosamine-4-sulfatase
- r4S
recombinant humanN-acetylgalactosamine-4-sulfatase
- PBS
phosphate buffered saline
- BME
basal modified Eagle's medium
- FBS
fetal bovine serum
- GalNAc4S-GlcA-GalitolNAc4S
-(N-acetyl-d-galactosamine-4-sulfate)-(1–4)--d-glucuronic acid)-(1–3)-N-acetyl-d-[1-3H]galactosaminitol-4-sulfate
- DS
dermatan sulfate
- MPS
mucopolysaccharidosis 相似文献
3.
Ion transport by rabbit colon 总被引:7,自引:0,他引:7
Raymond A. Frizzell Marilyn J. Koch Stanley G. Schultz 《The Journal of membrane biology》1976,27(1):297-316
Summary Descending rabbit colon, stripped ofmuscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO3 secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit currentI
sc
is determined solely by the rate of active Na transport and that the net movements of Cl and HCO3 are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO3 secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxesJ
sm
i
of Na, K and Cl suggest thatJ
sm
Na
,J
sm
K
and one-third ofJ
sm
Cl
may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such thatP
KP
NaP
Cl=1.00.070.11. Electrolyte transport byin vitro rabbit colon closely resembles that reported fromin vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms. 相似文献
4.
Summary The angular dependence of1JC,H in model compounds related to -linked oligosaccharides has been established by FPT INDO quantum chemical calculations. Values calculated for models of (1 1)-, (1 2)-, (1 3)- and (1 4)-linked disaccharides were compared, and the effect of the orientation of HO-2 elucidated. The angular dependence of1JC,H on the torsional angles H and H and the solvent dielectric constant (s) was characterized in the form:1JC,H = A cos2+B cos + C sin2 + D since + E + Fe. The1JC,H values, measured by DEPT methods for C-1-H-1 and C-X-H-X in cellobiose, cyclic trisaccharide and hexopyranoses were used to adjust the calculated angular dependences. Based on the occurrence of the conformers for agarobiose, neoagarobiose, mannobiose and methyl -xylobioside, the thermodynamically averaged <1JC,H > values were calculated. The results obtained (<1JC-1,H-1 > 162.4, <1JC-4, H-4 > 147.6 Hz for methyl -xylobioside; <1JC-1,H-1 > 162.4 and <1JC-4,H-4] > 147.6 Hz for mannobiose; <1JC-1,H-1 > 162.8 Hz for neo agarobiose and <1JC-1,H-1 > 163.2 Hz for agarobiose) agree well with the experimental values of 162.7, 147.5, 160.4, 147.2, 160.9 and 165.7 Hz, respectively. 相似文献
5.
Philip R. Steinmetz Russell F. Husted Allan Mueller Renaud Beauwens 《The Journal of membrane biology》1981,59(1):27-34
Summary The coupling between H+ transport (J
H) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.J
H was measured as the short-circuit current after Na+ transport was abolished with ouabain and by pH stat titration. The media were gassed with N2 and 1% CO2 (PO2<0.5 mm Hg) and contained 10mm glucose. Under these conditions,J
H was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (J
lac) of 47 bladders was plotted as a function of simultaneously measuredJ
H. The slope ofJ
lac onJ
H was 0.58±0.12 with an intercept forJ
lac atJ
H=0 of 0.55 mol/hr. Values for J
lac/J
H were determined in groups of individual bladders whenJ
H was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of J
lac/J
H indicates a high degree of coupling betweenJ
H andJ
lac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the J
lac/J
H values can be used to estimate the stoichiometry of H+ translocation. The movement of slightly less than 2 H+ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H+ flow in the proteolipid portion of H+-translocating ATPases. DCCD inhibited anaerobicJ
H without change in J
lac/J
H or basalJ
lac and, therefore, acted primarily on the H+ pump.S addition of vanadate also inhibitedJ
H, but the inhibition was associated with an increase inJ
lac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H+-ATPase characteristics that differ from mitochondrial ATPase in that H+ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H+ and lactate, the H+/ATP stoichiometry of the pump is about 2. 相似文献
6.
Michael E. Duffey Stephen M. Thompson Raymond A. Frizzell Stanley G. Schultz 《The Journal of membrane biology》1979,50(3-4):331-341
Summary Intracellular chloride activities, (Cl)
c
, and the electrical potential difference across the mucosal membrane, mc, were determined in the isolated small intestine of the winter flounder, using Cl-selective and conventional (KCl-filled) microelectrodes. In the presence of a Na-containing buffer mc averages –69mV and (Cl)
c
averages 24mM, a value that is 3.4 times that predicted for an equilibrium distribution across the mucosal membrane. On the other hand, when the tissue is then perfused with Na-free buffer, (Cl)
c
slowly falls to a value that does not differ significantly from that predicted for an equilibrium distribution, and mc depolarizes significantly. Finally, when the tissue is again bathed in the Na-containing buffer, (Cl)
c
rapidly returns to a value well above equilibrium.These results, together with those of Frizzellet al. (J. Membrane Biol.
46:27, 1979), provide direct evidence that: (1) Cl is accumulated against its electrochemical potential difference (32mV) by this tissue, and (2) this accumulation is coupled to and energized by the entry of Na down its steep electrochemical potential difference. 相似文献
7.
Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK
1=0.67±0.16 m,k
2=1.6±0.7 sec–1,k
–2=0.17±0.09 sec–1,K
1=6.3±1.7 m,k
2=9±4 sec–1 andk
–2=7±3 sec–1. The apparent dissociation constants of chloride from band 3,K
Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3. 相似文献
8.
A simple and accurate method is described for measurement of 1
J
CN splittings in isotopically enriched proteins. The method is of the quantitative J correlation type, and the 1
J
CN splitting is derived from the relative intensity in two 3D TROSY-HNCO spectra with 1
J
CN dephasing intervals of 1/(21
J
CN) (reference intensity) and 1/1
J
CN (residual intensity). If the two spectra are recorded under identical conditions and with the same number of scans, the random error in the 1
J
CN value extracted in this manner is inversely related to the signal-to-noise (S/N) in the reference spectrum. A S/N of 30:1 in the reference spectrum yields random errors of less than 0.2 Hz in the extracted 1
J
CN value. Dipolar couplings obtained from the difference in 1
J
CN splitting in the isotropic and liquid crystalline phase for the C-terminal domain of calmodulin are in excellent agreement with its 1.68-Å crystal structure, but agree considerably less with the 2.2-Å structure. 相似文献
9.
Summary The fluorescence enhancement of 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) upon binding to membranes was used to examine proximal tubule stilbene binding sites. Equilibrium binding studies of DBDS to renal brush border (BBMV) and basolateral membrane vesicles (BLMV) were performed using a fluorescence enhancement technique developed for red blood cells (A.S. Verkman, J.A. Dix and A.K. Solomon,J. Gen. Physiol.
81:421–449, 1983). In the absence of transportable anions, DBDS bound reversibly to a single class of sites on BLMV isolated from rabbit (K
d
=3.8 m) and rat (3.2 m); 100 m dihydro-4,4-diisothiocyano-2,2-disulfonic stilbene (H2DIDS) blocked >95% of binding. H2DIDS inhibitable DBDS binding was not detected using rat or rabbit BBMV. In rabbit BLMV, DBDSK
d
doubled with 10mm SO4, 50mm HCO3 and 100mm Cl, but was not altered by Na or pH (6–8). In stopped-flow experiments the exponential time constant for DBDS binding slowed with SO4, HCO3 and Cl, but was unaffected by Na. These results are consistent with competitive binding of DBDS and anions at an anion transport site. To relate DBDS binding data to anion transport inhibition we used35SO4 uptake to characterize several modes of rabbit BLM anion transport: H/SO4 and Na/SO4 cotransport, and Cl/SO4 countertransport. Each transport process was electroneutral and was inhibited by H2DIDS, furosemide, probenecid, chlorothiazide and DBDS. The apparentK
t
's for DBDS (3–20 m) were similar toK
d
for DBDS binding. These studies define a class of anion transport sites on the proximal tubule basolateral membrane measureable optically by a fluorescent stilbene. 相似文献
10.
Summary Ammonium (NH
4
+
) transport was investigated in Nostoc muscorum ISU (wild type) and spontaneous mutants resistant to cyanophage N-1 (Nm/N-1), streptomycin (Nm/Sm) and methylamine (Nm/MA). N2-fixing wild-type cells transported NH
4
+
via two transport systems: the high-affinity (K
m 11 M) and low-affinity (K
m 66 M), which formed 10 and 50-fold concentration gradients, respectively. The high-affinity system of Nm/MA (K
m 11 M) was similar to the wild type but the low-affinity system had reduced affinity for NH
4
+
(K
m 125 M), while Nm/N-1 and Nm/Sm mutants had only a high-affinity transport system (K
m 20 and 28 M, respectively). The growth of mutant Nm/N-1 was more sensitive to 1 mM NH
4
+
or methylamine than other strains, and also glutamine-synthetase activity was most reduced in NH
4
+
-grown cells. l-methionine-d, l-sulfoximine (20 M) treatment of N2-grown Nm/N-1 cells resulted in a higher rate of NH
4
+
efflux. The apparent alterations in kinetic constants of NH
4
+
transport in mutants and glutamine synthetase activity suggested that NH
4
+
in N. muscorum is transported by specific carrier(s) and the transport is genetically controlled. 相似文献
11.
Santiago LLovera Ramon Bonet Maria Dolores Simon-Pujol Francisco Congregado 《Applied microbiology and biotechnology》1993,39(3):424-426
The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe2+ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg2+, 20 m Ca2+ and 3.6 m Mn2+. Chromate reduction was not affected by the addition of MgCl2, CdCl2, ZnCl2, MnCl2, Na2SO4 (1000 m), and Na2MoO4 (100 m) to the activity assays. However, activity was inhibited by the presence of Na2SO4 (10 mm), Na2MoO4 (200 m) and ferric citrate. 相似文献
12.
The cellular model of short chain fatty acid stimulation of electroneutral Na-Cl absorption in large intestine proposes that SCFA, following its uptake across the apical membrane, recycles and is coupled to functional Na-H and Cl-short chain fatty acid exchanges. To establish the presence of a Cl-butyrate exchange (used as a model short chain fatty acid), studies of 36Cl and 14C-butyrate uptake across apical membrane vesicles of rat distal colon were performed. An outward butyrate-gradient stimulated transient accumulation of 36Cl uptake that was not inhibited by pH clamping with valinomycin (a K ionophore) and FCCP (a proton ionophore). Outward butyrate-gradient-stimulated 36Cl uptake was inhibited by 4,4-diisothiocyanatostilbene2,2-disulfonic acid (DIDS) with a half-maximal inhibitory concentration (IC50) of 68.4 m, and was saturated by both increasing extravesicular Cl concentration (K
m
for Cl of 26.8 ±3.4 mm and a V
max of 12.4±0.6 nmol/mg protein·9 sec) and increasing intravesicular butyrate concentration (K
m
for butyrate of 5.9 mm and a V
max for Cl of 5.9 nmol/mg protein · 9 sec). 36Cl uptake was also stimulated by outward gradients of other short chain fatty acids (e.g., propionate, acetate and formate). In contrast, an outward Cl gradient failed to enhance 14C-butyrate uptake. Extravesicular Cl more than extravesicular butyrate enhanced 36Cl efflux from apical membrane vesicles. These studies provide compelling evidence for the presence of an electroneutral, pH-activated, Cl-butyrate exchange which in concert with Na-H exchange is the mechanism by which butyrate stimulates electroneutral Na-Cl absorption.Abbreviations used AMV
apical membrane vesicles
- BLMV
basolateral membrane vesicles
- DIDS
4,4-diisothiocyanatostilbene 2,2-disulfonic acid
- FCCP
carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone
- MES
1-[N-morpholino]ethanesulfonic acid
- NMG
N-memyl-d-glucamine
- SCF
Ashort chain fatty acid
This study was supported in part by a Public Health Service research Grant (DK 14669) provided by the National Institute of Diabetes, Digestive and Kidney Diseases. Ms. Mary Guidone provided excellent secretarial assistance. 相似文献
13.
A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC
fast protein liquid chromatography
- NeuAc
5-N-acetyl-d-neuraminic acid
- 9-amino-NeuAc
5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid
- 9-acetamido-NeuAc
5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid
- 9-benzamido-NeuAc
5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid
- 9-fluoresceinyl-NeuAc
9-fluoresceinylthioureido-NeuAc
- 5-formyl-Neu
5-formyl--d-neuraminic acid
- 5-aminoacetyl-Neu
5-aminoacetyl--d-neuraminic acid
- CMP-NeuAc
cytidine-5-monophospho-N-acetylneuraminic acid
- GM1
Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide
- ST
sialyltransferase
- DTE
1,4-dithioerythritol
Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1. 相似文献
14.
N. Ito K. Nishi M. Nakajima Y. Okamura T. Hirota 《Histochemistry and cell biology》1988,89(2):121-128
Summary Effects of -galactosidase (from green coffee beans) digestion on lectin staining were examined in formalin-fixed, paraffin-embedded human pancreatic tissues from individuals of blood-group B and AB. Digestion with the enzyme resulted in almost complete loss of Griffonia simplicifolia agglutinin I-B4(GSAI-B4) staining in the acinar cells with concomitant appearance of Ulex europaeus agglutinin-I(UEA-I) staining in the corresponding cells. In addition, reactivity with soybean agglutinin(SBA) was also imparted by the enzyme digestion in GSAI-B4 positive acinar cells. -Galactosidase digestion following -galactosidase digestion neither reduced the reactivity with SBA nor induced the reactivity with Griffonia simplicifolia agglutinin-II(GSA-II) in GSAI-B4 positive cells, while in UEA-I positive cells, both reduction of SBA reactivity and appearance of GSA-II reactivity occurred after simple -galactosidase digestion as well as sequential digestion with - and -galactosidase. However, when -l-fucosidase digestion procedure was inserted between - and -galactosidase digestion, UEA-I staining imparted by -galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B4 positive acinar cells. Furthermore, after sequential digestion with -galactosidase and fucosidase, reactivity with peanut agglutinin(PNA) was revealed in GSAI-B4 positive acinar cells as well as UEA-I positive cells in secretors. In non-secretors, strong PNA staining was usually observed in the acinar cells throughout the glands without enzyme digestion. These results confirmed that the -galactosidase induced GSA-II reactivity and the fucosidase induced PNA reactivity are due to precursors of different kinds of blood-group determinants and suggest that at least two kinds of B antigen determinants, i.e. Gal(1-3)[Fuc(1-2)]Gal(1-3,4)GlcNac and Gal(1-3)-[Fuc(1-2)]Gal(1-3)GalNAc are produced in GSAI-B4 positive acinar cells. The synthesis of the latter type of B antigen is assumed to be controlled under the secretory gene in human pancreas.Abbreviation GalNAc
N-acetyl-d-galactosamine
- Gal
d-galactose
- GlcNAc
N-acetyl-d-glucosamine
- Fuc
l-fucose
- NeuNAc
N-acetylneuraminic acid (sialic acid) 相似文献
15.
Summary Models for active Cl transport across epithelia are often assumed to be universal although they are based on detailed studies of a relatively small number of epithelia from vertebrate animals. Epithelial Cl transport is also important in many invertebrates, but little is known regarding its cellular mechanisms. We used short-circuit current, tracer fluxes and ion substitutions to investigate the basic properties of Cl absorption by locust hindgut, an epithelium which is ideally suited for transport studies. Serosal addition of 1mm adenosine 35-cyclic monophosphate (cAMP), a known stimulant of Cl transport in this tissue, increased short-circuit current (I
sc) and net reabsorptive36Cl flux (J
net
Cl
) by 1000%. Cl absorption did not exhibit an exchange diffusion component and was highly selective over all anions tested except Br. Several predictions of Na- and HCO3-coupled models for Cl transport were tested: Cl-dependentI
sc was not affected by sodium removal (<0.05mm) during the first 75 min. Also, a large stimulation ofJ
net
Cl
was elicited by cAMP when recta were bathed for 6 hr in nominally Na-free saline (<0.001 to 0.2mm) and there was no correlation between Cl transport rate and the presence of micromolar quantities of Na contamination. Increased unidirectional influx of36Cl into rectal tissue during cAMP-stimulation was not accompanied by a comparable uptake of22Na.J
net
Cl
was independent of exogenous CO2 and HCO3, but was strongly dependent on the presence of K. These results suggest that the major fraction of Cl transport across this insect epithelium occurs by an unusual K-dependent mechanism that does not directly require Na or HCO3. 相似文献
16.
Lars-Åke Fransson Birgitta Havsmark Katsukiyo Sakurai Sakaru Suzuki 《Glycoconjugate journal》1992,9(1):45-55
To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated withp-hydroxyphenyl--d-xylopyranoside and [3H]galactose, and free [3H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After125I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on theN-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-Å, Havsmark B, Silverberg I (1990)Biochem J
269:381–8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.Abbreviations GAG
glycosaminoglycans
- CS
chondroitin sulfate
- DS
dermatan sulfate
- Ser
serine
- Xyl
d-xylose
- Gal
d-galactose
- GlcA
d-glucuronic acid
- IdoA
l-iduronic acid
- GalNAc
N-acetyl-d-galactosamine
- GlcNAc
N-acetyl-d-glucosamine
- HexA
4-deoxy-l-threo-hex-4-enopyranosyluronic acid
- HO-Phe
p-hydroxyphenyl group
- HO-Phe-Xyl
p-hydroxyphenyl-O--d-xylopyranoside
- O2N-Phe-Xyl
p-nitrophenyl--d-xylopyranoside
- OSO3
ester sulfate
- PAGE
polyacrylamide gel electrophoresis
- HPLC
high performance liquid chromatography
- FPLC
fast performance liquid chromatography
- LC
standard liquid chromatography 相似文献
17.
Summary The inhibitory effect of various stilbene disulfonates was examined on the swelling-activated Cl-dependent K transport (K-Cl cotransport) in low K sheep erythrocytes. Both diisothiocy-anatostilbenes H2DIDS and DIDS were found to be potent inhibitors. The DIDS concentration yielding 50% inhibition (IC50) of KCl cotransport was 60
m in the absence of external K and 3
m at physiological K concentration. Other stilbene derivatives, such as SITS (4-acetamido-4 isothiocyanatostilbene-2,2-disulfonic acid), were only effective in the presence of external K, whereas DNDS (4,4-dinitrostilbene-2,2-disulfonic acid) and ISA (4-sulfophenyl isothiocyanate) had only slight effects at a concentration of 1 mm. The augmenting effect of external K is due to a second K site, distinguishable from the K transport site by its much higher affinity. No inhibition occurred in the absence of external Cl, whether or not external Rb(K) was present. Additionally, DIDS inhibited K-Cl cotransport activated by thiol alkylation with N-ethylmaleimide (NEM) as well as by Mg depletion in the presence of A23187 and a chelator. We conclude that allosteric sites affect the stilbene binding. When these sites are saturated, changes in external K or Cl concentration do not affect the affinity for DIDS (noncompetitive inhibition).This work was supported by grants in aid from the American Heart Association. 相似文献
18.
Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz1H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU
National formulary unit
- COSY
correlation spectroscopy
- HOHAHA
homonuclear Hartmann-Hahn
- 1D or 2D
one- or two-dimensional
- IdoA
l-iduronic acid
- GlcA
d-gluco-4-enepyranosyluronic acid
- Di-0S
GlcA1-3GalNAc
- Di-4S
GlcA1-3GalNAc(4-sulfate)
- Di-4S
GlcA1-3GalNAc(4-sulfate)
- Di-6S
GlcA1-3GalNAc(6-sulfate)
- Di-6S
GlcA1-3GalNAc(6-sulfate)
- Di-diS
d
GlcA(2-sulfate)1-3GalNAc(6-sulfate)
- Di-diSE
GlcA1-3GalNAc(4, 6-disulfate)
- U
G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively 相似文献
19.
Akira Ono Shin-ichi Tate Yoshiharu Ishido Masatsune Kainosho 《Journal of biomolecular NMR》1994,4(4):581-586
Summary [13C5]-2-Deoxy-d-ribose, synthesized from [13C6]-d-glucose (98% 13C), was coupled with thymine to give [1,2,3,4,5-13C5]-thymidine (T) in an 18% overall yield. The thymidine was converted to the 3-phosphoramidite derivative and was then incorporated into a dodecamer 5-d(CGCGAATTCGCG)-3 by solid-phase DNA synthesis. Preparation of 0.24 mole of the labeled dodecamer, which is sufficient for a single NMR sample, consumed only 25 mg of glucose. By virtue of the 13C labels, all of the 1H-1H vicinal coupling constants in the sugar moieties were accurately determined by HCCH-E.COSY. 相似文献
20.
Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl2 buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P
Na:P
Ba:P
Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side. 相似文献