Biomedical subjects

L G Reddy

Publications and source records attributed to L G Reddy.

At least 19 recordsLinked to original sources

PubMed2001

Role of cysteine residues in structural stability and function of a transmembrane helix bundle.

To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare alpha-amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB.

Alanine↗

PubMed1999

Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study.

Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by beta-adrenergic stimulation. The mobility of PLB on SDS-PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, the oligomeric structure of PLB must be determined in its native milieu, a lipid bilayer containing the Ca-ATPase. Here we have used fluorescence energy transfer (FET) to study the oligomeric structure of PLB in SDS and dioleoylphosphatidylcholine (DOPC) lipid bilayers reconstituted in the absence and presence of Ca-ATPase. PLB was labeled, specifically at Lys 3 in the cytoplasmic domain, with amine-reactive fluorescent donor/acceptor pairs. FET between donor- and acceptor-labeled subunits of PLB in SDS solution and DOPC lipid bilayers indicated the presence of PLB oligomers. The dependence of FET efficiency on the fraction of acceptor-labeled PLB in DOPC bilayers indicated that it is predominantly an oligomer having 9-11 subunits, with approximately 10% of the PLB as monomer, and the distance between dyes on adjacent PLB subunits is 0.9 +/- 0.1 nm. When labeled PLB was reconstituted with purified Ca-ATPase, FET indicated the depolymerization of PLB into smaller oligomers having an average of 5 subunits, with a concomitant increase in the fraction of monomer to 30-40% and a doubling of the intersubunit distance. We conclude that PLB exists primarily as an oligomer in membranes, and the Ca-ATPase affects the structure of this oligomer, but the Ca-ATPase binds preferentially to the monomer and/or small oligomers. These results suggest that the active inhibitory species of PLB is a monomer or an oligomer having fewer than 5 subunits.

Calcium-Binding Proteins↗

PubMed1999

Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure.

Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by beta-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt-PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25 degrees C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25 degrees C, has no inhibitory activity when assayed at 25 degrees C. However, at 37 degrees C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37 degrees C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase.

Adenosine Triphosphate↗

PubMed1999

A fluorescence energy transfer method for analyzing protein oligomeric structure: application to phospholamban.

We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, was used to determine the dependence of FET on the number of subunits, the distance between labeled sites on different subunits, and the fraction of subunits remaining monomeric. By measuring FET as a function of the donor/acceptor ratio, the above parameters of the oligomeric structure can be resolved over a substantial range of their values. We used this approach to investigate the oligomeric structure of phospholamban (PLB), a 52-amino acid protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB regulates the SR Ca-ATPase. Because PLB exists primarily as a homopentamer on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, this hypothesis must be tested by determining directly the oligomeric structure of PLB in the lipid membrane. To accomplish this goal, PLB was labeled at Lys-3 in the cytoplasmic domain, with two different amine-reactive donor/acceptor pairs, which gave very similar FET results. In detergent solutions, FET was not observed unless the sample was first boiled to facilitate subunit mixing. In lipid bilayers, FET was observed at 25 degrees C without boiling, indicating a dynamic equilibrium among PLB subunits in the membrane. Analysis of the FET data indicated that the dye-labeled PLB is predominantly in oligomers having at least 8 subunits, that 7-23% of the PLB subunits are monomeric, and that the distance between dyes on adjacent PLB subunits is about 10 A. A point mutation of PLB (L37A) that runs as monomer on SDS-PAGE showed no energy transfer, confirming its monomeric state in the membrane. We conclude that FET is a powerful approach for analyzing the oligomeric structure of PLB, and this method is applicable to other oligomeric proteins.

Animals↗

PubMed1999

An autoinhibitory peptide from the erythrocyte Ca-ATPase aggregates and inhibits both muscle Ca-ATPase isoforms.

We have studied the effects of C28R2, a basic peptide derived from the autoinhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oligomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from skeletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphorescence anisotropy (TPA) was used to determine changes in the distribution of Ca-ATPase among its different oligomeric species in SR. C28R2, at a concentration of 1-10 microM, inhibits the Ca-ATPase activity of both skeletal and cardiac SR (CSR). In skeletal SR, this inhibition by C28R2 is much greater at low (0.15 microM) than at high (10 microM) Ca2+, whereas in CSR the inhibition is the same at low and high Ca2+. The effects of the peptide on the rotational mobility of the Ca-ATPase correlated well with function, indicating that C28R2-induced protein aggregation and Ca-ATPase inhibition are much more Ca-dependent in skeletal than in CSR. In CSR at low Ca2+, phospholamban (PLB) antibody (functionally equivalent to PLB phosphorylation) increased the inhibitory effect of C28R2 slightly. Fluorescence of fluorescein 5-isothiocyanate-labeled SR suggests that C28R2 stabilizes the E1 conformation of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2. After the addition of PLB antibody, C28R2 still stabilizes the E2 conformational state of CSR. Therefore, we conclude that C28R2 affects Ca-ATPase activity, conformation, and self-association differently in cardiac and skeletal SR and that PLB is probably not responsible for the differences.

Animals↗

PubMed1998

Co-reconstitution and co-crystallization of phospholamban and Ca(2+)-ATPase.

Significant advances have recently been made in understanding the regulation of Ca(2+)-ATPase by phospholamban and in modeling their structures. However, these insights would be furthered by determining the 3-D structure of both proteins within the membrane, thus revealing the structural basis for their interaction. To this end, we have developed methods for reconstituting purified Ca(2+)-ATPase with recombinant phospholamban. After reconstitution at high lipid-to-protein ratios, we have verified their functional association by measuring calcium transport and ATPase activity. Furthermore, we have grown co-crystals after reconstitution at low lipid-to-protein ratios. The structure of Ca(2+)-ATPase has recently been solved by cryoelectron microscopy at 8-A resolution, thus revealing transmembrane alpha-helices. Using a variety of constraints, we have associated these helices with the predicted transmembrane sequences to produce a detailed model for the packing of transmembrane helices. Structure determination of the co-crystals is currently underway, which we hope will eventually reveal the interaction of phospholamban with Ca(2+)-ATPase at a similar level of detail.

Animals↗

PubMed1998

Direct spectroscopic detection of molecular dynamics and interactions of the calcium pump and phospholamban.

In order to test molecular models of cardiac calcium transport regulation, we have used spectroscopy to probe the structures, dynamics, and interactions of the Ca pump (Ca-ATPase) and phospholamban (PLB) in cardiac sarcoplasmic reticulum (SR) and in reconstituted membranes. Electron paramagnetic resonance (EPR) and phosphorescence of probes bound to the Ca pump show that the activity of the pump is quite sensitive to its oligomeric interactions. In cardiac SR, PLB aggregates and inhibits the pump, and both effects are reversed by PLB phosphorylation. Previous analyses of PLB's oligomeric state were only in detergent solutions, so we used EPR and fluorescence to determine the oligomeric structure of PLB in its native state in lipid bilayers. Wild-type PLB is primarily oligomeric in the membrane, while the mutant L37A-PLB is monomeric. For both proteins, phosphorylation shifts the dynamic monomer-oligomer equilibrium toward oligomers, and induces a similar structural change, as indicated by tyrosine fluorescence; yet L37A-PLB is more effective than wild-type PLB in inhibiting and aggregating the pump. Fluorescence energy transfer shows that the Ca pump increases the fraction of monomeric PLB, indicating that the pump preferentially binds monomeric PLB. These results support a reciprocal aggregation model for Ca pump regulation, in which the Ca pump is aggregated and inhibited by association with PLB monomers, and phosphorylation of PLB reverses these effects while decreasing the concentration of PLB monomers. To investigate the structure of the PLB pentamer in more detail, we measured the reactivities of cysteine residues in the transmembrane domain of PLB, and recorded EPR spectra of spin labels attached to these sites. These results support an atomic structural model, based on molecular dynamics simulations and mutagenesis studies, in which the PLB pentamer is stabilized by a leucine-isoleucine zipper within the transmembrane domain.

Animals↗

PubMed1997

Analysis of homo- and heterodimerization of retinoid receptors in solution.

To characterize the dimerization of retinoid receptors in solution, RAR alpha homodimers and RAR alpha-RXR alpha heterodimers, formed in the absence or the presence of a naturally occurring RA response element (betaRARE) under different ionic conditions, were analyzed by size-exclusion fast protein liquid chromatography and sucrose density gradient sedimentation. In the presence of [3H]RA both RAR alpha and RXR alpha existed primarily as monomers of 50 kDa in solutions containing 80 mM KCl. However, when betaRARE was included in these incubations, a 40-fold increase in the occurrence of both the RAR alpha homodimers and the RAR alpha-RXR alpha heterodimers (125 kDa) was observed. The presence of RAR alpha and RXR alpha in the betaRARE-associated homo- and heterodimers was confirmed by the positive interaction of the receptors with the specific antibodies. Both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers bound betaRARE even in the absence of the ligand RA with the heterodimer showing a 2- to 4-fold greater affinity than the homodimer for the DNA binding element. When the receptors were incubated in solutions of increasing ionic concentration (50-300 mM KCl), a decrease in the amount of both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers was accompanied by a corresponding increase in the monomeric fraction even in the presence of betaRARE, suggesting that the high salt concentrations inhibit the surface to surface interactions between the monomers. These observations suggest that in vivo, as in solution, the formation of a stable retinoid receptor dimer complex is dependent upon both receptor-receptor and receptor-RARE interactions.

Animals↗

PubMed1997

How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase.

In an attempt to better define the parameters governing reconstitution and two-dimensional crystallization of membrane proteins, we have studied Ca2(+)-ATPase from rabbit sarcoplasmic reticulum. This ion pump forms vanadate-induced crystals in its native membrane and has previously been reconstituted at high lipid-to-protein ratios for functional studies. We have characterized the reconstitution of purified Ca2(+)-ATPase at low lipid-to-protein ratios and discovered procedures that produce long, tubular crystals suitable for helical reconstruction. C12E8 (n-dodecyl-octaethylene-glycol monoether) was used to fully solubilize various mixtures of lipid and purified Ca2(+)-ATPase, and BioBeads were then used to remove the C12E8. Slow removal resulted in two populations of vesicles, and the proteoliposome population was separated from the liposome population on a sucrose density gradient. These proteoliposomes had a lipid-to-protein ratio of 1:2, and virtually 100% of molecules faced the outside of vesicles, as determined by fluorescein isothiocyanate labeling. Cycles of freeze-thaw caused considerable aggregation of these proteoliposomes, and, if phosphatidyl ethanolamine and phosphatidic acid were included, or if the bilayers were doped with small amounts of C12E8, vanadate-induced tubular crystals grew from the aggregates. Thus our procedure comprised two steps-reconstitution followed by crystallization-allowing us to consider mechanisms of bilayer formation separately from those of crystallization and tube formation.

Animals↗

PubMed1996

Characterization of cellular retinoic acid-binding protein I from chick embryo and its ligand binding properties.

In order to characterize cellular retinoic acid-binding protein (CRABP) from chick embryos, we resolved the [3H]RA-binding activity of embryo extracts by Mono Q anion-exchange chromatography. Fractions containing [3H]RA-binding proteins eluted in two peaks at 7 and 10 min with the latter showing 10-fold higher activity than the former. When the [3H]RA-binding activity in the major peak was resolved on a Superose-12 size-exclusion column, a protein of about 15,000 Da, similar in size to CRABP I or II, was eluted. The identity of this RA-binding component as CRABP I was confirmed by its immunopositive reaction with a CRABP I-specific monoclonal antibody. The chick embryo CRABP I, upon electrophoresis on native gel, however, showed slower migration than the mouse CRABP I, although both exhibited similar isoelectric pH (pI) of about 4.5. Equilibrium binding studies performed under saturating levels of RA indicated that the retinoid bound to the chick CRABP I with a Kd of 27 nM, a value similar to that reported for the native form of this protein from other species. Moreover, as indicated by their IC50 values, the relative binding affinities of various RA analogs for chick CRABP I are consistent with those obtained with human and mouse CRABP I. These results demonstrate that the major RA-binding protein expressed in chick embryo, while having a different charge as judged by electrophoretic mobility, is similar to mouse CRABP I in its size, pI, antigenic specificity, and ligand binding properties.

Animals↗

PubMed1996

Analysis of the effects of CRABP I expression on the RA-induced transcription mediated by retinoid receptors.

The involvement of cellular retinoic acid-binding protein I (CRABP I) in the RA signaling was investigated by examining its effects on the interaction of retinoid receptors (RARs and RXRs) with RA-response elements (RAREs) as well as on the RA-induced transcription mediated by retinoid receptors. Analysis of the expression of mouse CRABP I from a cDNA expression plasmid in COS-1 cells revealed that this protein was about 5-fold more abundant in cytosol than in nuclei. The identity and the localization of CRABP I in the cytoplasm as well as the nuclei were also confirmed by the immunoperoxidase staining of the transfected COS-1 cells with CRABP I-specific antibody. When the nuclear extract containing a 10-fold molar excess of CRABP I was incubated with RAR alpha extract in the presence of [3H]RA and resolved on an FPLC size-exclusion column, a 20% decrease in the bound radioactivity in the RAR alpha fraction was accompanied by a proportional increase in the CRABP I fraction. In contrast, the addition of CRABP I did not significantly affect the interaction of RAR alpha or RAR alpha-RXR alpha heterodimers with RAREs. Moreover, the coexpression of CRABP I in CV-1 cells did not markedly inhibit or enhance the transcription activated by RARs and RAR alpha-RXR alpha heterodimers under RA concentrations ranging from 10(-10) to 10(-6) M. These results demonstrate that CRABP I, while it might be important for RA homeostasis, is not directly involved in the retinoid receptor-mediated RA-signaling pathway.

Animals↗

PubMed1996

Purified, reconstituted cardiac Ca2+-ATPase is regulated by phospholamban but not by direct phosphorylation with Ca2+/calmodulin-dependent protein kinase.

Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to beta-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (approximately 1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by approximately 50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on Vmax for calcium transport.

Adenosine Triphosphate↗

PubMed1995

Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca(2+)-ATPase.

Phospholamban (PLB) is a small, transmembrane protein that resides in the cardiac sarcoplasmic reticulum (SR) and regulates the activity of Ca(2+)-ATPase in response to beta-adrenergic stimulation. We have used the baculovirus expression system in Sf21 cells to express milligram quantities of wild-type PLB. After purification by antibody affinity chromatography, the function of this recombinant PLB was tested by reconstitution with Ca(2+)-ATPase purified from skeletal SR. The results obtained with recombinant PLB were indistinguishable from those obtained with purified, canine cardiac PLB. In particular, PLB reduced the apparent calcium affinity of Ca(2+)-ATPase but had no effect on Vmax. At pCa 6.8, PLB inhibited both calcium uptake and ATPase activity of Ca(2+)-ATPase by 50%. This inhibition was fully reversed by addition of a monoclonal antibody to PLB, which mimics the physiological effects of PLB phosphorylation. Maximal PLB regulatory effects occurred at a molar stoichiometry of approximately 3:1, PLB/Ca(2+)-ATPase. We also investigated peptides corresponding to the two main domains of PLB. The membrane-spanning domain, PLB26-52, appeared to uncouple ATPase hydrolysis from calcium transport, even though the permeability of the reconstituted vesicles was not altered. The cytoplasmic peptide, PLB1-31, had little effect, even at a 300:1 molar excess over Ca(2+)-ATPase.

Animals↗

PubMed1995

Secondary structure and orientation of phospholamban reconstituted in supported bilayers from polarized attenuated total reflection FTIR spectroscopy.

We have studied the secondary structure of native phospholamban (PLB), a 52-residue integral membrane protein that regulates calcium uptake into the cardiac sarcoplasmic reticulum, as well as its 27-residue carboxy-terminal transmembrane segment (PLB26-52). The relative contents of alpha-helix, beta-strand, and random coil, as well as the spatial orientations of the alpha-helices of these molecules, reconstituted in dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer membranes, were determined using polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. The major component of the amide I' bands of PLB and PLB26-52 was centered at 1654-1657 cm-1 and was assigned to alpha-helix. The fraction of alpha-helix in native PLB was 64-67% (33-35 residues), and the transmembrane peptide PLB26-52 contained 73-82% alpha-helix (20-22 residues); small fractions of beta- and random structures were also identified. The orientational order parameter (S) of the alpha-helical component of PLB26-52 in DMPC was S = 0.86 +/- 0.09, indicating that the transmembrane helix was oriented approximately perpendicular to the membrane plane. Assuming the transmembrane domain of PLB resembles the peptide PLB26-52, the additional alpha-helical residues in PLB were assigned to the cytoplasmic helix and determined to have an order parameter S = -0.15 +/- 0.30. This may imply that the cytoplasmic helix was tilted from the membrane normal by an angle of 61 +/- 13 degrees or, alternatively, may indicate a wide angular distribution.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Immobilized nucleases.

Nucleases are important analytical enzymes and are used widely for the determination of nucleic acid structure. Their application depends on the specificity and mode of action of the particular enzyme. Nucleases have also found application in the production of flavor enhancers like 5' IMP and 5' GMP, removal of nucleic acids in single cell protein preparations, and as therapeutic agents. Immobilization of nucleases and the use of immobilized nucleases for various biotechnological applications have been reviewed.

Biotechnology↗

PubMed1991

A new retinoid-binding protein with a low molecular weight.

Cellular retinoid-binding proteins and nuclear receptors may mediate the intracellular transport and the action of retinoids in the control of differentiation and tumorigenesis. We report a new retinoid-binding protein (Ret BP) with a molecular size of 4,000 that binds retinol, retinoic acid, and some of their derivatives. Purification of Ret BP from chick skin cytosol involved DEAE-Sephadex, Sephadex G-100, and Mono Q column chromatography. The Ret BP-retinoid complex eluted at 195 mM NaCl during Mono Q column chromatography using a 0-300 nM NaCl gradient. Superose-12 column chromatography indicated a molecular size of 4,000 for Ret BP. The binding protein showed a pI of 6.8 on electrofocusing in ampholines of pH 3-10. Ret BP may act as an affinant for retinoids in the cell, and may serve to dispense the ligands to their respective functionally active sites.

Animals↗

PubMed1990

Isolation, partial purification and characterization of nuclear retinoic acid receptors from chick skin.

Nuclear receptors (RARs) for retinoic acid (RA) are considered to be the ultimate mediators of the action of RA in the control of cell differentiation and inhibition of tumorigenesis. We have isolated and partially purified and characterized RAR from a RA-responsive tissue, chick embryo skin. The purification steps included Affi-Gel blue chromatography, ultrafiltration, size exclusion chromatography, and preparative isoelectric focusing. The electrofocusing of RAR-[3H]RA complex in ampholines (pH 3-10) revealed that the receptors have an isoelectric pH of 7.5. Whereas pronase-digested the RAR-[3H]RA complex completely, DNase showed 20-35% and RNase showed negligible digestive action on the complex. The ligand binding to RAR was completely inhibited by a mercury compound. RAR-alpha- and RAR-beta-specific antibodies, on Western blot analysis, immunoreacted with a protein having a molecular weight of 50,000, presumably RAR. Binding affinity studies revealed that biologically active analogs of RA with a free COOH group (e.g., 13-cis-RA, RO-13-7410, Ch 55, and Am 80) showed, like RA, high binding affinity for RAR, whereas biologically ineffective analogs of RA (e.g., furyl and pyridyl) were poor binders. Other groups of retinoids, in which the COOH group was either lacking or blocked, did not bind to RAR whether or not they were biologically active.

Animals↗

PubMed1989

Preparation and properties of RNase T2 immobilized on concanavalin A-sepharose.

Partially purified RNase T2 (EC 2.7.7.17) from Aspergillus oryzae was bound through its carbohydrate moiety to Concanavalin A-Sepharose. The retention of activity was high, ranging from 70% at low enzyme load to approximately 9% at high enzyme load. Though there was no change in the pH and temperature optima, the pH stability and the Km decreased after immobilization. Compared to the soluble enzyme, the immobilized RNase T2 showed enhanced temperature stability and more resistance to metal ions. Both soluble and immobilized enzymes were stable to 8 M urea. On repeated use, the bound enzyme retained more than 60% of its initial activity after six cycles.

Aspergillus oryzae↗