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A Sivaprasadarao

Publications and source records attributed to A Sivaprasadarao.

At least 19 recordsLinked to original sources
PubMed1998

Expression, characterization and engineered specificity of rat epididymal retinoic acid-binding protein.

Epididymal retinoic acid-binding protein (ERABP) is the major androgen-dependent protein present in the lumen of the epididymis and is thought to be involved in sperm maturation. It displays a high degree of three-dimensional structural similarity to serum retinol-binding protein (RBP). Although both proteins interact with retinoids, RBP exhibits a broad specificity, binding retinol, retinoic acid and retinaldehyde with roughly equal affinities, whereas ERABP is specific for all-trans- and 9-cis-retinoic acids. Consistent with this, the binding pockets of the two proteins are different: in RBP it is predominantly hydrophobic, whereas that for ERABP is amphipathic, with a network of charged residues at the open end of the binding pocket. In order to investigate the roles of these charged residues, Arg-80 and Glu-63 have been mutated to isoleucine. The resultant double mutant, Glu-63-->Ile/Arg-80-->Ile, as well as the wild-type protein, were subsequently expressed in Escherichia coli as fusion proteins, with the streptavidin recognition sequence (Strep) tagged to their C-termini. The expressed proteins were purified in a single step by streptavidin-affinity chromatography and their ligand-binding properties were examined using fluorimetric titrations. Whereas the wild-type ERABP binds only retinoic acid, the double mutant is capable of binding retinol, retinoic acid and retinaldehyde with similar affinities. These observations provide experimental support for the proposition that the charged residues near the open end of the binding pocket are responsible for restricting the specificity of ERABP for retinoic acid. These studies demonstrate that changes in specificity can be engineered into lipocalins.

Animals↗
PubMed1998

The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor.

The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated. The experimental system consisted of the [3H]retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes. [3H]Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin. Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP. The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP. The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism. Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.

Humans↗
PubMed1998

Adenosine receptor mRNA levels during postnatal renal maturation in the rat.

Adenosine may affect the pattern of intrarenal blood flow during renal development. It provides an angiogenic stimulus for the growth of new blood vessels and may be involved in compensatory renal growth. It is therefore of interest to investigate the expression of adenosine receptor genes during postnatal renal development. In the present study this was carried out by measuring adenosine receptor mRNA levels in rats aged between 2 and 60 days. The order of abundance of adenosine receptor mRNA levels in 60-day-old rats was A2A > A2B > or = A1 > A3. A1 receptor mRNA levels showed only small changes with increasing age although, by contrast, A3 receptor mRNA increased markedly with age with levels at 60 days twenty-fold greater than at 2 days. A2A receptor mRNA levels declined during renal maturation with transcript numbers four- to fivefold that at 12-18 days compared with numbers at 60 days. By contrast to the A2A receptor, there were no significant changes in the renal levels of A2B receptor mRNA during kidney maturation. During postnatal renal maturation, the levels of mRNA for A2A and A3 adenosine receptor subtypes undergo marked changes which may be related to functional maturation, morphological development, or both.

Animals↗
PubMed1997

Renal adenosine A1 receptor binding characteristics and mRNA levels during the development of acute renal failure in the rat.

1. The binding characteristics and mRNA levels for renal adenosine A1 receptors were investigated in normal rats and rats with acute renal failure (ARF) induced by either glycerol or HgCl2. 2. Saturation isotherms determined from the binding of [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), a selective adenosine A1 antagonist, to renal membranes of untreated rats gave values of 0.62 nM for the equilibrium dissociation constant (Kd) and 19.9 fmol mg-1 protein for the density of binding sites (Bmax). No saturable binding was observed with [3H]-2-(p-(carboxylethyl)-phenylethylamino)-5'-N-ethylcar box amido adenosine ([3H]-CGS 21680), a selective adenosine A2a agonist. 3. By contrast to time-matched controls, renal membranes obtained from rats 16 and 48 h following the induction of ARF with glycerol, showed statistically significant increases (2-4 fold) in both Bmax and Kd for the binding of [3H]-DPCPX. No significant changes in the binding characteristics of [3H]-DPCPX were noted with membranes from rats 48 h following the production of ARF with HgCl2. 4. Adenosine A1 receptor mRNA levels were significantly elevated 0.5, 16 and 48 h following induction of ARF with glycerol, whilst no change was noted in mRNA levels for beta-actin at the same time points. No statistically significant changes in adenosine A1 receptor or beta-actin mRNA levels were noted 48 h after the induction of ARF with HgCl2. 5. This study indicates that glycerol-induced ARF in the rat is associated with an increase in renal adenosine A1 receptor density which appears to result from increased transcription of the gene for this receptor. An increase in adenosine A1 receptor density in renal resistance vessels may explain, at least in part, the enhanced renal vasoconstrictor response to adenosine in glycerol-induced ARF that was noted in a previous study.

Acute Kidney Injury↗
PubMed1996

Membrane receptor for odour-binding proteins.

Specific binding of 125I-labelled bovine odour-binding protein (OBP) to isolated membranes from nasal mucosa was demonstrated. The interaction reached equilibrium within 30 min at 37 degrees C and was reversible. A Scatchard analysis of the equilibrium binding revealed a single population of binding sites, with the calculated equilibrium dissociation constant and maximum number of binding sites being 2.25 +/- 0.5 microM and 18.5 +/- 2 pmol/mg of membrane protein respectively (n = 2). Receptor activity was decreased on digestion by trypsin, proteinase K or endoglycosidase H, was heat labile and was sensitive to thiol-group-specific reagents. With the exception of rat and mouse major urinary proteins, which exhibit a high degree of structural similarity with OBP and bind similar ligands, other members of the lipocalin family, such as retinol-binding protein and beta-lactoglobulin, failed to inhibit the binding of 125I-labelled OBP to its receptor. The receptor seems not to be restricted to olfactory tissues, as it was detected in a variety of other tissues. This suggests that OBP is unlikely to play a role only in olfactory signal transduction. It might have a much broader role within the body; possibilities include a role in detoxification or signalling.

Animals↗
PubMed1995

A beta-subclass phosphatidylinositol-specific phospholipase C from squid (Loligo forbesi) photoreceptors exhibiting a truncated C-terminus.

A PCR-based strategy has been used to isolate a full length cDNA encoding a phosphatidylinositol-specific phospholipase C from a sized cDNA squid (Loligo forbesi) retinal library. The predicted protein sequence contains 875 amino acids, with calculated M(r) 98,181, and has marked similarity with PLC beta-isoforms, including conservation of the 'X' and 'Y' regions. It is unique in having a major C-terminal truncation. A major protein of apparent M(r) 120,000 estimated by SDS-PAGE has been isolated from squid photoreceptors and identified by partial protein sequence analysis to correspond to the protein sequence predicted from the cDNA clone. This protein has been shown to hydrolyse phosphatidylinositol 4,5-bisphosphate. It is not yet clear whether this represents the major light-activated PLC in squid vision.

Amino Acid Sequence↗
PubMed1994

Changes in activation gating of IsK potassium currents brought about by mutations in the transmembrane sequence.

Expression of the rat kidney IsK protein in Xenopus oocytes produces slowly-activating potassium channel currents. We have investigated the relationship between structure and function of the single putative membrane-spanning domain using site-directed mutagenesis. Six mutants were constructed in which consecutive individual amino acids (53 to 58) of the transmembrane region were substituted by cysteine. Expression of four of these mutants in Xenopus oocytes resulted in currents which were similar to wild-type. However, for one mutant (position 55) activation curves were shifted in a hyperpolarising direction and for another mutant (position 58) activation curve were shifted in a depolarising direction. This suggests that the hydrophobic phenylalanine residues at positions 55 and 58 may play a critical role in IsK activation gating. This spacing of functional amino acids at every third residue may indicate an alpha-helical conformation for the membrane-spanning domain of IsK. Furthermore, these results also indicate that one face of the helix may represent a region of subunit association.

Amino Acid Sequence↗
PubMed1994

Solubilization and purification of the retinol-binding protein receptor from human placental membranes.

The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.

Binding Sites↗
PubMed1994

Structure-function studies on human retinol-binding protein using site-directed mutagenesis.

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.

Base Sequence↗
PubMed1994

Modulation by protein kinase A of a cloned rat brain potassium channel expressed in Xenopus oocytes.

A potassium channel from rat brain was expressed in Xenopus oocytes in order to study modulation of channel function by phosphorylation via protein kinase A. Application of 8-Br-cAMP to oocytes expressing the drk1 channel (with the first 139 amino acids of the N terminus deleted, delta Ndrk1) caused a voltage-independent elevation of current amplitude, which was not seen for endogenous currents or for wild-type full-length drk1 channel. This effect on delta Ndrk1 was blocked by pre-injection of oocytes with Walsh-peptide protein kinase A inhibitor, suggesting mediation via protein kinase A. The protein kinase inhibitor also reduced both delta Ndrk1 and full-length drk1 currents. Substitution of the serine residues by alanine at one or both of the two consensus protein kinase A phosphorylation sites on the C terminus (residues 440 and 492) of delta Ndrk1 resulted in a loss of function of the expressed channels. These results indicate that phosphorylation via protein kinase A modulates drk1 channel function and that both consensus phosphorylation sites seems to be essential for channels to function.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector.

In order to express human serum retinol-binding protein (sRBP) in Escherichia coli in a form that is structurally indistinguishable from the native protein, we placed the coding sequence of the RBP cDNA next to that of the outer membrane protein A (OmpA) signal sequence in the secretion vector, pIN-III-OmpA1. However, this construct did not generate detectable expression of RBP in E. coli. When the DNA fragment consisting of the ribosome-binding site and the OmpA-RBP fusion sequence was subcloned downstream to the T7 promoter of pKS-Bluescript, however, the resultant construct (pOmp-RBP2) gave low but detectable secretion of RBP into the periplasm. Deletion of the 3' untranslated region of the RBP cDNA (pOmp-RBP3) further improved the expression (by approx. 20-fold). After charging with retinol, the secreted RBP was purified from the periplasm on a transthyretin-affinity resin. The purified protein exhibited all the three molecular recognition properties characteristic of sRBP, i.e. it interacted with retinol, transthyretin and its cell-surface receptor. Comparison of the receptor binding properties of the recombinant RBP (rRBP) with those of the serum protein revealed that while the affinity of rRBP is similar to sRBP (50 +/- 20 nM), the Bmax of the rRBP is about 6-8-fold higher. This indicates that a major proportion of RBP, isolated from serum, is incapable of interacting with the receptor.

Autoradiography↗
PubMed1988

The interaction of retinol-binding protein with its plasma-membrane receptor.

125I-labelled retinol-binding protein (RBP) bound to specific receptors in human placental brush-border membranes. Binding at 22 degrees C reached equilibrium within 15 min, but prolonged incubation caused a subsequent decline. Scatchard analysis of the equilibrium binding data at 22 degrees C and 15 min showed high-(3.0 +/- 2.7 x 10(-9) M) and low-(9.5 +/- 3.5 x 10(-8) M) affinity binding components. 125I-RBP, bound to membranes at 22 degrees C for 15 min and subsequently dissociated with excess unlabelled RBP, exhibited biphasic dissociation kinetics consisting of fast and slow components of release. In contrast, Scatchard analysis and dissociation kinetics of the binding that had taken place at 37 degrees C for 1 h showed the fast-dissociating/low-affinity binding component, but little of the slow-dissociating/higher-affinity binding component. When 125I-RBP, after incubation with membranes at 37 degrees C for 1 h, was re-isolated and subjected to dissociation kinetic analysis using a fresh batch of membranes, the fast-dissociating phase was unchanged, but the slow phase was almost absent. The complex kinetics were interpreted in terms of a heterogeneity in RBP consisting of high- and low-affinity binding forms. The higher-affinity-binding form is thought to be converted into the lower-affinity state on binding to the receptor. Transthyretin inhibited 125I-RBP binding to the membrane, suggesting that free, rather than transthyretin-associated, RBP bound to the receptor. The RBP receptor was trypsin-, heat- and thiol-group-specific-reagent sensitive and was highly specific for RBP.

Binding, Competitive↗
PubMed1988

The mechanism of uptake of retinol by plasma-membrane vesicles.

The mechanism of retinol uptake by human placental brush-border membrane vesicles was investigated using initial-velocity studies of [3H]retinol uptake from the [3H]retinol-RBP (retinol-binding protein) complex. The process was rapid and time- and temperature-dependent. The uptake was specifically reversed by the addition of native or apo-RBP, but not by serum albumin. By contrast, uptake of free [3H]retinol was temperature-independent, partially reversible and showed no requirement for a specific protein for reversibility. Treatment of membrane vesicles with p-chloromercuribenzenesulphonate (PCMBS), which inhibited 125I-RBP binding, also inhibited the uptake of retinol from RBP, but the uptake of free retinol was unaffected. Addition of PCMBS after the attainment of steady-state uptake equilibrium abolished the binding of RBP, but did not affect the retinol already taken up from RBP. The results suggest that binding of RBP to its specific receptor is obligatory for the subsequent delivery of retinol to the membrane. Since the studies were carried out on isolated membrane vesicles, endocytosis of RBP is most unlikely to be involved in the placental transport of retinol. A double-reciprocal plot of initial velocity versus [3H]retinol-RBP concentration gave an apparent Km of 116 +/- 13 nM. Transthyretin decreased the rate of uptake of [3H]retinol from RBP without substantially altering the steady-state uptake levels, suggesting that membranes take up retinol from uncomplexed RBP. High-pressure gel-filtration chromatography showed that [3H]retinol is largely transferred to a membrane component with an apparent molecular mass of 125 kDa.

4-Chloromercuribenzenesulfonate↗
PubMedDate not supplied

Measurement of the movement of the S4 segment during the activation of a voltage-gated potassium channel.

Voltage-gated ion channels contain a positively charged transmembrane segment termed S4. Recent evidence suggests that depolarisation of the membrane potential causes this segment to undergo conformational changes that, in turn, lead to the opening of the channel pore. In order to define these conformational changes in structural terms, we have introduced single cysteine substitutions into the S4 segment of the prototypical Shaker K+ channel at various positions and expressed the mutants in Xenopus oocytes. The cells were depolarised to induce K+ currents and the effect of application of 100 microM parachloromercuribenzenesulphonate (PCMBS) on these currents was examined by the two-electrode voltage-clamp technique. PCMBS inhibited K+ currents elicited by mutants L358C, L361C, V363C and L366C, but not those by V367C and S376C. Since PCMBS is a membrane-impermeable cysteine-modifying reagent, the data suggest that depolarisation must have caused the S4 segment to move out of the lipid bilayer into the extracellular phase rendering the residues at positions 358, 361, 363 and 366 susceptible to PCMBS attack. The lack of effect of PCMBS on V367C suggests that the exposure of S4 terminates at L366. Detailed analysis of L361C mutant revealed that the S4 movement can occur even below the resting potential of the cell, at which potential voltage-gated K+ channels are normally in a non-conducting closed state.

4-Chloromercuribenzenesulfonate↗