Pedro Cuatrecasas

[31] Affinity chromatography

Publisher Summary The selective isolation and purification of enzymes and other biologically important macromolecules by “affinity chromatography” exploits the unique biological property of the proteins or polypeptides to bind ligands specifically and reversibly. Affinity chromatography exploits the phenomenon of specific biological interaction in a large variety of protein–ligand systems. A solution containing the macromolecule to be purified is passed through a column containing an insoluble polymer or gel to which a specific competitive inhibitor or other ligand has been covalently attached. Proteins not exhibiting appreciable affinity for the ligand pass unretarded through the column, whereas those which recognize the inhibitor are retarded in proportion to the affinity existing under the experimental conditions. The specifically adsorbed protein can be eluted by altering the composition of the solvent so that dissociation occurs. Affinity chromatography may be useful in concentrating dilute solutions of proteins, in removing denatured forms of a purified protein, and in the separation and resolution of protein components resulting from specific chemical modifications of purified proteins. Inherent advantages of this method of purification are the rapidity and ease of a potentially single-step procedure, the rapid separation of the protein to be purified from inhibitors and destructive contaminants, such as proteases, and protection from denaturation during purification by active site ligand-stabilization of protein tertiary structure.

Multiple opiate receptors

Abstract The discovery of opiate receptors has been followed by the exciting discovery of endogenous opiate-like substances; the enkephalins and endorphins. These opioid peptides bind to opiate receptors with high affinity and mimic some of the effects of morphine itself. They have been postulated to act as neurotransmitters or neuromodulators in regulating pain perception, locomotor activity, body temperature, behaviour, and neuroendocrine functions. The discovery of enkephalin has also led to the discovery of multiple types of opiate receptor and in this article Chang, Hazum and Cuatrecasas discuss whether the complex effects of opioid peptides are mediated by single or multiple types of opiate receptors.

Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin

Thrombin rapidly induces the formation of labeled phosphatidic acid from platelets prelabeled with [17C]arachidonate or 32PO34- and specifically decreases by 50--75% the content of phosphatidylinositol. Ionophore A23187 also stimulates phosphatidate labeling, but less effectively than thrombin. This effect on phosphatidic acid is blocked by increasing the levels of cyclic AMP by preincubation with dibutyryl cyclic AMP, cyclic AMP-phosphodiesterase inhibitors or prostacyclin. Indomethacin and eicosatetraynoic acid do not alter the production of phosphatidate, indicating independence from cyclooxygenase or lipoxygenase products. Increased turnover of [14C]- or [32P]phosphatidate occurs within 2--5 s after platelet activation by thrombin and is observed before endogenous, 14C-labeled arachidonate can be detected. The rate of phosphatidate formation parallels the induced rate of serotonin release. Release of [3H]serotonin is not affected by eicosatetraynoic acid. Phosphatidate production reflects the generation of diacylglycerol by C-type phospholipase degradation of phosphatidylinositol. Diacylglycerol and phosphatidic acid may participate in the membrane modification related to the early changes in platelet shape, release reactions or aggregation which occur on stimulation.

Regulation of arachidonate metabolism via lipoxygenase and cyclo-oxygenase by 12-HPETE, the product of human platelet lipoxygenase.

Abstract Human platelets metabolize arachidonic acid via lipoxygenase and cyclo-oxygenase. The labile lipoxygenase product, 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), stimulates its own production by increasing lipoxygenase activity. However, 12-hydroxy-eicosatetraenoic acid (12-HETE), the end-product of the lipoxygenase pathway in human platelets, does not share this activity. In addition, 12-HPETE strongly suppresses prostaglandin and thromboxane production by inhibiting platelet cyclo-oxygenase. These effects of arachidonic acid hydroperoxides exhibit isomeric specificity in platelet homogenates; 12-HPETE is more potent than 11-HPETE, which is more potent than 9- and 8-HPETE. Collagen- and arachidonate-induced platelet aggregation is blocked by 12-HPETE.

The phosphatidylinositol cycle and the regulation of arachidonic acid production

An increase in the metabolism of phosphatidylinositol occurs in a wide variety of tissues by the action of specific ligands1–3. In platelets, the interaction of thrombin with its receptor initiates the degradation of phosphatidylinositol by the action of a specific phospholipase C (refs 4–8). In normal conditions of stimulation, the resultant 1,2-diacylglycerol is rapidly and completely phosphorylated to phosphatidic acid4–11. The formation of phosphatidic acid precedes the release of arachidonic acid from the phospholipids of stimulated platelets5. This early appearence of phosphatidate might result in the initial production of arachidonic acid and lysophosphatidic acid by the action of a phospholipase A2 specific for phosphatidate12. Phosphatidate/lysophosphatidate could induce calciumgating13–15 and subsequently stimulate phospholipases of the A2-type8, that degrade phosphatidylcholine, phosphatidyl-ethanolamine and a further fraction of phosphatidylinositol6. Alternatively, the lysophosphatidate produced may serve as a substrate for the transfer of arachidonate directly from other phospholipids16,17 to form new phosphatidate which in turn can release more arachidonate. Overall, such a sequence would be equivalent to phospholipase A2 activation of other phospholipids. Our present data indicate that when the release of arachidonic acid is completely inhibited by cyclic AMP or quinacrine, phosphatidic acid is redirected entirely to phosphatidylinositol and there is no production of arachidonate. In these conditions, the availability of calcium might be profoundly restricted. The correlation in platelets of a phosphatidylinositol by a specific phospholipase A2 might suggest that these phenomena are applicable to activations in other cell systems.

Peptide Hormone-Induced Receptor Mobility, Aggregation, and Internalization

ONE of the basic challenges facing modern biomedical research is the understanding of how hormones produce chemical signals that cause alterations in cellular metabolism. Peptide hormones induce a ...

Cyclic adenosine 3′,5′-monophosphate and prostacyclin inhibit membrane phospholipase activity in platelets

Abstract [ 14 C]-Arachidonic acid is incorporated mainly into phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of horse platelet membranes. Treatment of washed platelets with thrombin leads to a rapid loss of radioactivity from these phospholipids. The liberated [ 14 C]-arachidonate is immediately transformed into hydroxyacids and thromboxanes. Treatment with dibutyryl cyclic AMP, cyclic AMP phosphodiesterase inhibitors or prostacyclin, a newly discovered prostaglandin that stimulates platelet adenylate cyclase, prevents the action of thrombin on phospholipid break-down as well as on platelet aggregation. Dibutyryl cyclic AMP does not affect the metabolism of exogenous [ 14 C]-arachidonic acid. Cyclic AMP may thus play a crucial role in the regulation of platelet phospholipase acitivity, and this could explain at least in part the inhibition of aggregation caused by substances which, like prostacyclin, raise the levels of cyclic AMP.

[6] Topics in the methodology of substitution reactions with agarose

Publisher Summary Agarose beads have been one of the most useful solid supports in affinity chromatography. This chapter describes various alternative methods that can be used to activate agarose for the covalent attachment of small ligands and proteins. In addition, some general problems encountered in the use of such derivatives are presented and possible solutions are suggested. Nearly all methods for coupling ligands or proteins to agarose depend on the initial modification of the gel with CNBr. An alternative method that is rapid, simple, and safe and which promises to result in chemically stable ligand-agarose bonds has been developed. The method depends on the oxidation of cis-vicinal hydroxyl groups of agarose by sodium metaperiodate (NalO 4 ) to generate aldehyde functions. These aldehydic functions react at pH 4–6 with primary amines to form Schiff bases; these are reduced with sodium borohydride (NaBH 4 ) to form stable secondary amines. The reductive stabilization of the intermediate Schiff base is best achieved with sodium cyanoborohydride (NaBH 3 CN) because this reagent, at a pH between 6 and 6.5, preferentially reduces Schiff bases without reducing the aldehyde functions of the agarose. Because sodium cyanoborohydride selectively reduces only the Schiff bases, it shifts the equilibrium of the reaction to the right and thereby drives the overall reaction to completion.

Transferrin receptor: its biological significance

ConclusionsThe TR is a vital surface component which has been demonstrated to be involved in processes critical for cell metabolism and growth. This review has attempted to briefly touch on the more well understood aspects of study of the TR. These aspects include the biochemical characterization of the TR and the functional studies concerning the central role of the TR in binding transferrin for the purpose of internalization and accumulation of intracellular iron. Other less well-understood and controversial aspects surrounding our present knowledge of the TR have been highlighted and discussed. These include: the nature of the biochemical signal involved in triggering receptor endocytosis; the role for the transferrin-TR interaction or the TR alone in regulation of cellular growth processes; and the possible clinical role(s) for the TR in anti-tumor therapy.

Estimation of hormone receptor affinity by competitive displacement of labeled ligand: Effect of concentration of receptor and of labeled ligand

Abstract The concentration of ligand (e.g., hormone) at which a given fraction of bound labeled ligand is competitively displaced from its binding site (e.g., receptor) depends upon the concentration of labeled ligand and of binding sites as well as on the affinity of the ligand. The ligand concentration at which 50% of the bound, labeled ligand is displaced [K′d(app)] is often taken (mistakenly) to be equal to the dissociation constant of the ligand. For an ideal bimolecular reactions, it is shown that K′d(app) actually exceeds the dissociation constant by an amount equal to the labeled ligand concentration plus the binding site concentration minus three halves the concentration of labeled ligand that would be bound in the absence of unlabeled ligand. For both epidermal growth factor and insulin, the concentration of unlabeled hormone at which a given fraction of bound labeled hormone is displaced from placenta membranes is increased by increasing the labeled hormone concentration or the placenta membrane concentration. K′d(app) will give spuriously high estimates of the dissociation constants for these hormones if measured at high labeled hormone or binding site concentrations. These considerations also have important implications for comparative studies of receptors in different species or metabolic states, and for the feasibility of “radioreceptor” assays.