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The relationship between neurotransmitter metabolite concentrations and measurements of ventricular size on CAT scans and pneumoencephalographs was investigated in 15 patients with severe affective disorder. An association was identified between reduced levels of plasma free tryptophan and ventricular enlargement and also between raised ventricular CSF levels of 5 HIAA and ventricular enlargement.

General Ligand Affinity Chromatography: N6-(6-Aminohexyl) 3',5'-ADP Sepharose as an Affinity Adsorbent for the CoA-Dependent Enzyme, Succinate Thiokinase

Publisher Summary This β -galactosidase system has played an important role in the development of the view that immobilized ligands with dissociation constants in the millimolar range are capable of promoting very strong specific adsorption, and this view in turn has led to the development of the concept of “progressively perpetuating effectiveness.” This concept is not consistent with much other evidence, or with the theoretical treatment summarized in the next section, and it is difficult to reconcile with kinetic theory in general. Again, the observations that largely gave rise to this concept are explainable in terms of nonbiospecific rather than biospecific adsorption.

The synthesis of a new coenzyme A analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-CoA, suitable for immobilisation through its terminal amino group to support matrices, is described. The synthetic route starts with bis(CoA) and involves the following steps: alkylation with iodoacetic acid and rearrangement yielding bis(N6-carboxymethyl-CoA), elongation of the carboxymethyl terminal with 1,6-diaminohexane using carbodiimide to yield bis(N6-[N-(6-aminohexyl)-carbamoylmethyl]-CoA) and finally the splitting of this bis[CoA analogue) through reduction with dithiothreitol to give the final product in approximately 10% overall yield. This CoA analogue showed 'coenzymic activity' with the enzymes acetyl-CoA synthetase, phosphotransacetylase and succinic thiokinase. Covalent binding of the CoA analogue to Sepharose 4B was normally carried out using its S-(5-thio-2-nitrobenzoic acid) derivative as this allows a convenient way for determining the amount of ligand coupled, based on the amount of 5-thio-2-nitrobenzoic acid liberated from the gel after reduction with dithiothreitol. After covalent binding of the CoA analogue to water-soluble activated dextran 70, the analogue was recycled while present in an ultrafiltration cell using the enzymes phosphotransacetylase and citrate synthase. The reaction was followed by measuring the citrate formed on addition of acetylphosphate and oxaloacetate. In affinity chromatographic studies it was shown that the CoA-Sepharose preparation could bind the CoA-dependent enzymes citrate synthase and succinic thiokinase and these could be biospecifically eluted using soluble CoA.

1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD+through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD+(probably through the 8 position of the adenine residue) to a number of different spacer-arm–agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD+derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD+. Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD+-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed.

Publisher Summary In the search for an effective immobilized ligand, attention was concentrated on analogs of pyruvate rather than of lactate, because kinetic studies indicate that pyruvate has considerably higher affinity for the enzyme. As explained in this chapter, the kinetic mechanism of the enzyme requires the addition of NADH to the irrigating buffer during affinity chromatography. Under these conditions catalytically susceptible immobilized analogs (e.g., β-mercaptopyruvate, attached through the β-thiol group) underwent enzymatic reduction when the enzyme was applied, and such analogs proved to be unsatisfactory for affinity chromatography. Oxamate is a structural analog of pyruvate that is not catalytically susceptible, but retains the binding characteristics of pyruvate for lactate dehydrogenase. This analog, immobilized through the amide grouping proved to be a very effective affinity chromatographic ligand. The amide nitrogen group corresponds to the methyl group of pyruvate and was chosen as the point of attachment because available specificity studies indicated that pyruvate analogs in which the methyl group is substituted remain effective as substrates for lactate dehydrogenase.