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R R Rando

Publications and source records attributed to R R Rando.

At least 55 records · Page 3Linked to original sources
PubMed1995

Photoaffinity labeling of retinoic acid-binding proteins.

Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues.

Affinity Labels↗
PubMed1995

Specific binding of aminoglycoside antibiotics to RNA.

BACKGROUND: Aminoglycoside antibiotics interfere with ribosomal protein synthesis and with intron splicing. Various lines of evidence suggest that RNA is the molecular target for aminoglycosides, but little is known about the recognition process. Is recognition of a particular aminoglycoside specific for certain RNA structures? If so, what are the rules for recognition? We have begun to investigate this problem by in vitro selection of RNA molecules that can specifically bind to the aminoglycoside antibiotic tobramycin. RESULTS: An RNA diversity library was used to select for sequences capable of binding to the aminoglycoside antibiotic tobramycin. After six cycles of selection, 82% of the RNA bound to tobramycin specifically. The selected RNA was reverse-transcribed into DNA, which was then cloned. At low selection stringency, an extremely large number of clones, on the order of 10(7), produced RNAs capable of binding tobramycin with Kds in the microM range (values similar to that observed for the binding of tobramycin to Escherichia coli ribosomes). Sequencing of 18 of the clones revealed no obvious consensus sequence. At higher selection stringencies (Kds in the nM range) only two consensus sequences for binding were observed. CONCLUSIONS: We have shown that RNA molecules can be readily selected that bind the aminoglycoside tobramycin. The RNAs that bind tobramycin with high affinity contain consensus binding regions that may be confined to predicted stem-loop structures. These studies open the way for understanding the basis of RNA-aminoglycoside recognition.

Anti-Bacterial Agents↗
PubMed1994

Novel methyltransferase activity modifying the carboxy terminal bis(geranylgeranyl)-Cys-Ala-Cys structure of small GTP-binding proteins.

Proteins containing CX3, CXC, and CC (where C is cysteine and X is undefined) undergo posttranslational isoprenylation at their cysteine residues. In the case of proteins which terminate in CX3, proteolytic removal of X3 is followed by the carboxymethylation of the isoprenylated cysteine residue. CXC proteins also undergo C-terminal methylation. The present study addresses the question of whether this methylation is catalyzed by a different isoprenylated protein methyltransferase than that previously described for CX3 proteins. The S-adenosylmethionine (AdoMet) dependent methylation of a small peptide-N-acetyl-S-geranylgeranyl-L-cysteinyl-L-alanyl-S-geranylgeranyl- L- cysteine (Ac(GG)CysAla(GG)Cys)--was investigated using membranes from a variety of bovine tissues as sources of enzyme. Ac(GG)CysAla(GG)Cys was a substrate for methylation, while Ac(GG)Cys(GG)Cys was not. Reciprocal inhibition studies on the methylation reactions of the CXC peptide and of N-acetyl-S-farnesyl-L-cysteine (AFC), a previously described methyltransferase substrate, suggested that these reactions are catalyzed by distinct enzymatic activities. Farnesylthioacetic acid (FTA), a potent competitive inhibitor of the methylation of AFC, did not inhibit the methylation of the CXC peptide. Moreover the KI values for S-adenosylhomocysteine and S-adenosylethionine inhibition differed for the two enzymatic activities. These data indicate that more than one AdoMet-dependent methyltransferase is involved in the carboxymethylation of isoprenylated proteins.

Amino Acid Sequence↗
PubMed1994

Thiol dependent isomerization of all-trans-retinoic acid to 9-cis-retinoic acid.

The important biological effector 9-cis-retinoic acid can be generated by liver microsomes or by bovine serum albumin in detergent. The mechanism of this isomerization reaction is the subject of these studies. The protein mediated isomerization process is shown to be thiol- and pH-dependent. Moreover, the retinoic acids are also isomerized by 1-dodecanethiol in the presence of detergents. This isomerization process is pH-dependent as well, with isomerization rates increasing with pH. The isomerization reactions are quenched with free radical traps, such as alpha-tocopherol and ascorbic acid, suggesting that a thiol radical mechanism, rather than a thiolate anion-dependent mechanism, is implicated here. The pH dependence can be understood in terms of a thiol radical mechanism, because thiol radicals are produced from thiolate anions in the presence of oxygen. The facile thiol-mediated isomerization of the retinoic acids suggests that this could be a physiologically relevant mechanism for the formation of 9-cis-retinoic acid from all-trans-retinoic acid.

Animals↗
PubMed1994

Functional significance of G protein carboxymethylation.

Heterotrimeric G proteins are isoprenylated and methylated on their gamma subunits. Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of control of G protein activity. A method for selectively demethylating isoprenylated and methylated G proteins is reported here using retinal transducin (T) as a model system. It was found that pig liver esterase is capable of completely hydrolyzing T beta gamma, but not T alpha beta gamma, to its unmethylated form. This allows for the direct determination of the activities of methylated and unmethylated T beta gamma. The activities of the T beta gamma s were determined by measuring their abilities to stimulate GTP-gamma-S exchange in the presence of T alpha and photoactivated rhodopsin (R*). It is reported here that, in detergent, unmethylated T beta gamma was at least as active as its methylated counterpart. Therefore, methylation does not affect the intrinsic ability of T beta gamma to functionally interact with T alpha and R*. However, in disk membranes an approximate 2-fold effect was observed, with the methylated T beta gamma being more efficient. Therefore, isoprenylated protein methylation may play a quantitative role in signal transduction, even though the intrinsic activities of the methylated subunits in detergent may be no different from their unmethylated counterparts. Finally, the use of pig liver esterase to demethylate isoprenylated proteins should allow for a clarification of the physiological role(s) of isoprenylated protein carboxymethylation in general.

Animals↗
PubMed1994

Farnesyl-L-cysteine analogs can inhibit or initiate superoxide release by human neutrophils.

A series of farnesylcysteine analogs was studied with respect to their abilities to interfere with fMet-Leu-Phe (fMLP)-stimulated superoxide (O2-.) release by human neutrophils. Simple acyl derivatives of farnesyl-L-cysteine, such as the N-acetyl (L-AFC) and N-isobutyryl derivatives (L-iBFC), which are substrates for the isoprenylated protein methyltransferase, can block O2-. release. The N-butyryl analog (L-BFC), which is an isomer of L-iBFC and also a substrate for the methyltransferase, does not inhibit O2-. release but actually stimulates it in the absence of fMLP. Other analogs, including the N-pivaloyl derivative, which has been found to be neither a substrate nor an inhibitor of methyltransferase, also stimulate very large quantities of O2-. production. The stimulatory effects of these derivatives are saturable and exquisitively sensitive to small structural changes in the analogs. The signal transduction pathway(s) utilized by pivaloyl derivatives for triggering O2-. generation is very similar to that employed by fMLP. These data make it clear that farnesyl-L-cysteine analogs do not produce their pharmacological effects in neutrophils via methyltransferase blockade. This could be further demonstrated by showing that sinefungin and S-adenosylhomocysteine, both powerful and general methyltransferase inhibitors which bind at the S-adenosylmethionine site, had no effect in preventing the increased oxygen consumption associated with O2-. production in permeabilized neutrophils. These studies reveal that farnesyl-L-cysteine analogs interact with a hitherto undefined target in neutrophils that may be exploited for inhibiting or stimulating the inflammatory or antimicrobial responses of these cells.

Chromatography, High Pressure Liquid↗
PubMed1994

Mechanistic studies on human platelet isoprenylated protein methyltransferase: farnesylcysteine analogs block platelet aggregation without inhibiting the methyltransferase.

The kinetic mechanism of the human platelet S-adenosyl-L-methionine (AdoMet)-linked isoprenylated protein methyltransferase was studied and determined to be ordered bibi. AdoMet binds first, and S-adenosyl-L-homocysteine (AdoHcy) departs last. Simple N-acetylated farnesylated cysteine analogs, such as N-acetyl-S-farnesyl-L-cysteine (AFC), are excellent substrates for the enzyme. Although many N-acetylated farnesylated cysteine analogs are excellent substrates for the enzyme, analogs with bulky moieties adjacent to the farnesylcysteine are neither substrates nor inhibitors of the enzyme. Two molecules of this class, N-benzoyl-S-farnesyl-L-cysteine (BzFC) and N-pivaloyl-S-farnesyl-L-cysteine (PFC) are useful in sorting out the putative physiological role of the methyltransferase in mediating human platelet aggregation because their pharmacological activities are unlinked to methyltransferase inhibition. When studied as inhibitors of platelet aggregation, the analogs are as active, or more active, than bona fide methyltransferase inhibitors of similar structure. Therefore, although it is possible that methyltransferase inhibitors, such as AFC, inhibit the enzyme when applied to cells, the observed pharmacological effects appear to be unrelated to this blockade. The new FC analogs described here have revealed a new signal transduction target which will be of some interest to explore.

Animals↗
PubMed1994

Isomerization of all-trans-retinoic acid to 9-cis-retinoic acid.

The discovery of the biological activity of 9-cis-retinoic acid raises questions as to its mode of biosynthesis. A simple mechanism involves the direct isomerization of all-trans-retinoic acid to 9-cis-retinoic acid. It is shown here that bovine liver membranes, but not supernatant fractions, can isomerize all-trans-retinoic acid into 9-cis-retinoic acid and 13-cis-retinoic acid. The concentration of 9-cis-retinoic acid generated approaches its equilibrium concentration, which is determined here to be approximately 15%. However, the isomerization process could not be shown to be saturable, and is first-order in all-trans-retinoic acid in the concentration range measured (8.3 nM to 3 microM). Isomerization reactions measured using bovine liver microsomes appear to be mediated by thiol groups, as they can be blocked by group-specific thiol-blocking reagents such as N-ethylmaleimide. It is interesting to note that the non-stereospecific behaviour observed here mimics what is observed when all-trans-retinoic acid is applied to cells. Finally, significant formation of 9-cis-retinoids was not found when the reaction was carried out with liver microsomes and either all-trans-retinol or all-trans-retinal.

Animals↗
PubMed1993

Endoproteolysis of non-CAAX-containing isoprenylated peptides.

A microsomal endoprotease specifically cleaves isoprenylated peptides of the CAAX motif, such as N-acetyl-S-all-trans-farnesyl-L-cysteine (AFC-VIM), at the isoprenylated cysteine residue. It is shown here that endoproteolysis will also occur with peptides which are not of the CAAX type. Peptide substrates modeled after the Delta virus large antigen carboxyl-terminus (CRPQ) are endoproteolytically hydrolyzed by liver microsomes. AFC-RPQ is hydrolyzed with a KM = 12.4 microM and a Vmax = 0.27 nmol/min/mg, and AGGC-RPQ is hydrolyzed with a KM = 7.9 microM and a Vmax = 0.042 nmol/min/mg. Moreover, a series of potent inhibitors of the endoproteolysis of AFC-AAX-containing peptides are ineffective at inhibiting the hydrolysis of AFC-RPQ and AGGC-RPQ, suggesting the existence of isoforms of the endoprotease.

Amino Acid Sequence↗
PubMed1993

Acyl group transfer from the sn-1 position of phospholipids in the biosynthesis of n-dodecyl palmitate.

The wax ester n-dodecyl palmitate is shown to be synthesized by retinal pigment epithelial membranes. The biosynthesis of this ester is phospholipid dependent and occurs via the transfer of a palmitoyl group from the sn-1 position of lecithin to n-dodecanol. When retinal pigment epithelial membranes are used as the source of enzyme, the apparent Michaelis constant for n-dodecanol in this process is 65.8 microM, and the maximal velocity for n-dodecyl palmitate synthesis is 16.2 nmol/(h.mg of protein). The enzymatic activity is membrane associated and shows a maximum velocity between pH8 and pH9. This transesterification process appears to be similar to the lecithin retinol acyl transferase reaction and is a further example of acyl group transfer reactions from the sn-1 position of phospholipids.

Acyl Coenzyme A↗
PubMed1993

Affinity labeling of lecithin retinol acyltransferase.

Lecithin retinol acyltransferase (LRAT) transfers acyl groups regiospecifically from the sn-1 position of lecithins to all-trans-retinol (vitamin A) and similar retinoids. LRAT is essential for the biosynthesis of 11-cis-retinal, the visual pigment chromophore. LRAT is also required for the general dietary mobilization of vitamin A. The enzyme is membrane-bound and has been solubilized and partially, but not completely, purified. It is demonstrated here that all-trans-retinyl alpha-bromoacetate (RBA) is a potent irreversible affinity labeling agent of LRAT. The measured KI = 12.1 microM and the pseudo-first-order rate constant for inhibition is kinh = 8.2 x 10(-4) s-1. The specificity of the inhibition process is further evidenced by the observation that alpha-bromoacetate derivatives of hydrophobic alcohols which are not substrates for LRAT, such as cholesterol and beta-ionol, are not inhibitors of the enzyme. Labeling of the partially purified enzyme with 3H-RBA showed a single radiolabeled band of molecular weight approximately 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Acyltransferases↗
PubMed1993

Inhibitors of the isoprenylated protein endoprotease.

The isoprenylation pathway requires an endoprotease that cleaves the modified protein at the isoprenylated cysteine residue. This endoprotease was readily assayed with simple tetrapeptide substrates of the type N-acetyl-S-farnesyl-L-Cys-(AFC)-Val-Ile-Met, where AFC and the tripeptide are the products of the hydrolysis. The endoprotease proved to be unaffected by (1) serine protease inhibitors, including (4-amidinophenyl)methanesulfonyl fluoride, aprotinin, and leupeptin, by (2) cysteine protease inhibitors, including E-64 and leupeptin [the enzyme is, however, inhibited by p-(hydroxymercuri)benzoate], by (3) metalloprotease inhibitors, including phosphoramidon, EDTA, and 1,10-phenanthroline, or by (4) the aspartyl protease inhibitor pepstatin. The conclusion from these data is that the enzyme is probably not a metalloenzyme. N-Boc-S-all-trans-farnesyl-L-cysteine (BFC) derivatives containing a statine moiety are also not inhibitory, strongly suggesting that the enzyme is not an aspartyl protease. However, the enzyme is potently inhibited by the aldehyde derivative of BFC (K1 = 1.9 microM), which is consistent with the idea that the enzyme is a serine or cysteine protease. Potent tetrapeptide-based competitive inhibitors were prepared. Analogs with the scissile bond modified so that hydrolysis could not occur were excellent inhibitors. An analog containing BFC-statine-Val-Ile-Met inhibited the endoprotease with a K1 = 64 nM. The equivalent pseudopeptide psi (CH2-NH) analog was almost as potent, indicating that the statine moiety simply represents a nonhydrolyzable linker.

Amino Acid Sequence↗
PubMed1993

Kinetic mechanism of lecithin retinol acyl transferase.

Lecithin retinol acyl transferase transfers acyl groups regiospecifically from the 1-position of lecithins to all-trans-retinol (vitamin A) and similar retinoids. LRAT is essential for the biosynthesis of 11-cis-retinal, the visual pigment chromophore, and is also required for the general dietary mobilization of vitamin A. The kinetic mechanism of this enzyme is described here, KM and Vmax values were determined for the substrates dipalmitoylphosphatidylcholine (DPPC) [1.38 microM and 0.17 microM/(min-mg), respectively] and for all-trans-retinol [0.243 microM and 0.199 microM/(min-mg), respectively]. In order to distinguish between a ping-pong bi-bi mechanism and a rapid equilibrium random or ordered bi-bi mechanism, the velocity of product formation as a function of one of the substrates at different fixed concentrations of the other substrate was measured. The parallel lines generated are entirely consistent with a ping-pong bi-bi mechanism in which DPPC first binds to LRAT and acylates it and rule out both simple random binding and ordered kinetic mechanisms. Further evidence for a ping-pong bi-bi mechanism comes from partial exchange reaction studies which show that LRAT can catalyze acyl group interchange between two different lecithin derivatives. Finally, the ping-pong reaction was established as being ordered, using the potent and reversible dead-end inhibitor 13-desmethyl-13,14-dihydro-all-trans-retinyl trifluoroacetate. This compound proved to be competitive with respect to DPPC, with a KI = 11.4 microM, and uncompetitive with respect to all-trans-retinol.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Substrate specificity of the isoprenylated protein endoprotease.

Proteins containing a CAAX motif at their carboxyl termini are subject to isoprenylation at the cysteine residue. Proteolytic trimming of isoprenylated proteins is essential in the activation of these proteins. A microsomal endopeptidase activity has been identified which cleaves all-trans farnesylated cysteine containing tetrapeptides between the modified residue and the adjacent amino acid to liberate the modified cysteine residue and an intact tripeptide. Structure/activity studies are reported here on this endopeptidase activity which are consistent with the premise that this protease is identical to the one normally involved in the cellular isoprenylation pathway. The protease only processes peptides which possess an isoprenyl moiety. Within the isoprenyl series, the enzyme hydrolyzes all-trans-farnesyl-, all-trans-geranylgeranyl-, and geranyl-containing peptides. The protease also recognizes the AAX sequence, because the protease behaves either stereospecifically or stereoselectively with respect to the individual amino acids of the tripeptide. The enzyme only measurably hydrolyzes isoprenylated peptides possessing L-amino acids at C and A. On the other hand, there is a small but measurable hydrolysis of isoprenylated peptides containing a D-amino acid at X.

Amino Acid Sequence↗
PubMed1992

A microsomal endoprotease that specifically cleaves isoprenylated peptides.

A microsomal enzymatic activity is described that can specifically cleave the tetrapeptide N-acetyl-S-farnesyl-L-Cys-L-Val-L-Ile-L-Ser between the isoprenylated cysteine residue and the valine residue. Km and Vmax values are measured as 5.8 microM and 251 pmol/min per mg of protein, respectively. Proteolytic cleavage of the substrate is stereospecific because the substitution of a farnesylated D-cysteine residue for the L-amino acid leads to the abolition of substrate activity. A free carboxyl-terminal group is also required for substrate activity because methyl esterification renders the substrate inert. The tripeptide N-acetyl-S-farnesyl-L-Cys-L-Val-L-Ile and the dipeptide N-acetyl-S-farnesyl-L-Cys-L-Val are also hydrolyzed by the protease. Again, stereospecificity is observed at the isoprenylated residue. Hydrolysis of the farnesylated tetrapeptide is not inhibited by a 5-fold excess of the nonfarnesylated tetrapeptide, suggesting that isoprenylation is important for substrate activity. This activity is probably the same as the proteolytic activity proposed to cleave isoprenylated proteins terminating in a Cys-Ali-Ali-Xaa motif, where Ali refers to aliphatic amino acid. These proteins include the ras family of G proteins and the heterotrimeric G proteins. Proteolytic maturation of these essential isoprenylated signal-transducing elements is a key step in their activation.

Amino Acid Sequence↗