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91.
92.
A.A. Bernardo F.T. Kear J.A. Stim O.S. Ruiz J.A.L. Arruda 《The Journal of membrane biology》1996,154(2):155-162
We have previously partially purified the basolateral Na+/HCO−
3 cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement
of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO−
3 cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity.
Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that
the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against
the Na+/HCO−
3 cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO−
3 cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes.
The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO−
3 cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of
the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence
of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared
with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies
recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in
the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and
small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO−
3 cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes
of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against
the 56 kDa basolateral protein inhibit the activity of the Na+/HCO−
3 cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact
at or near the substrate binding sites. The Na+/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues.
Received: 27 January 1996/Revised: 23 July 1996 相似文献
93.
Elizabeth A. Kingsley Teresa E. Carter Kevin D. Barrow Pamela J. Russell 《Cancer immunology, immunotherapy : CII》1996,41(6):348-354
A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR
F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway. 相似文献
94.
Zafar U. Khan Antonia Gutiérrez Celia P. Miralles Angel L. De Blas 《Neurochemical research》1996,21(2):147-159
Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ1, γ2S, γL, and γ3) have been made. The proportion of GABAA receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation.
In all tested regions of the rat brain, the γ1, and γ3 subunits are present in considerable smaller proportion of GABAA receptor than the γ2 subunit. Immunocytochemistry shows that γ1 immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate
gyrus, γ1 immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous
synapses of the perforant pathway. In contrast, γ3 immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are
also rich in γ2S. In the cerebellu, γ1 immunolabeling was localized on the Bergmann glia. The γ2S and γ2L subunits are differentially expressed in various brain regions. Thus the γ2S is highly expressed in the olfactory bulb and hippocampus whereas the γ2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization
and immunoprecipitation techniques have revealed. The γ2S and γ2L coexist in some brain areas and cell types. Moreover, the γ2S and γ2L subunits can coexist in the same GABAA receptor pentamer. We have shown that this is the case in some GABAA receptors expressed in cerebellar granule cells. These GABAA receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ1, γ2S, γ2L and γ3 subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in
the expression of the γ2S and γ2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the
GABAA receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms
are discussed in terms of the high molecular and binding heterogeneity of the native GABAA receptors in brain.
Special issue dedicated to Dr. Kinya Kuriyama 相似文献
95.
Gordon C. K. Roberts 《Neurochemical research》1996,21(9):1117-1124
NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The
kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in
drug metabolism. Cytochromes P450 play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the
haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate
complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance
to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme
A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined,
providing valuable information on the catalytic mechanism.
Special issue dedicated to Dr. Herman Bachelard. 相似文献
96.
Patrick J. Cummings Sharon S. Rowland Nancy E. Hooper Richard S. Schwalbe 《Microbiology and immunology》1996,40(11):883-886
Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications. 相似文献
97.
Hiroyuki Yamaguchi Hitoshi Miura Kazuoki Ohsumi Takako Osaki Haruhiko Taguchi Tomoko Yamamoto Tomoko Hanawa Sachio Ogata Shigeru Kamiya 《Microbiology and immunology》1996,40(1):77-80
To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3. 相似文献
98.
用菠菜甜菜碱醛脱氢酶 ( BADH)免疫巴比西 ( BALB/c)小鼠 ,将其脾细胞与骨髓瘤细胞 SP2 /O-Ag1 4融合 ,在 1 92孔中 ,有约 1 4 %孔生长的杂交瘤细胞 ,用间接酶联免疫方法 ( ELISA)检测表现为阳性。选择其中 2 G3和 2 D10 细胞系 ,用有限稀释法进行克隆化培养 ,约 2 0 %克隆化细胞为强阳性。选择其中 2 G3- H3细胞株注射到 BALB/c小鼠腹腔中诱导腹水 ,腹水的单抗效价为 1∶ 1 0 3。应用 BADH单抗检查了大麦、水稻、高粱、小麦幼苗的叶片和根的粗提物 ,均呈阳性反应 ,表明 BADH除在光合组织中存在外 ,在非光合组织中也可能存在。讨论了非光合组织 BADH的意义 相似文献
99.
Zhao Rongrui Wang Wenze Wu Bowei Hoebeke Johan Hjalmarson Åke Fu Michael L. X. 《Molecular and cellular biochemistry》1996,163(1):185-193
The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K+ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated Ek of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward Ica and outward Ik simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated ICa but not the isoprenaline-stimulated Ik. The antibody- or carbachol-induced outward K+ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated Ica were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events. 相似文献
100.
Alexander Schouten Jan Roosien Fred A. van Engelen G. A. M. de Jong A. W. M. Borst-Vrenssen Jacoline F. Zilverentant Dirk Bosch Willem J. Stiekema Fred J. Gommers Arjen Schots Jaap Bakker 《Plant molecular biology》1996,30(4):781-793
The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed. 相似文献