Physicochemical interactions in synthetic templates

Abstract

This thesis entitled “physicochemical interactions on synthetic templates” is divided into three sections. Since the unifying theme of the work is the use of affinity chromatographic techniques for the purification of different types of biological macromolecules, the thesis commences with a general review on this topic (Section?I). Section?II deals with the identification and purification of the gene II protein of coliphage and Section?III describes an immunocytochemical technique for the identification of strychnine?sensitive sites on neuronal synapses. Section?I: Affinity chromatography has made rapid inroads into several areas of the biological sciences and is an invaluable tool for the purification of macromolecules. In this section, the use of various matrices like agarose, dextrans, polyacrylamide and glass as inert supports for affinity chromatography has been discussed. Procedures of derivatizations for coupling of ligands have also been indicated. Sepharose is probably the ideal carrier for affinity chromatography, because of the high degree of substitution possible, ease of activation and the loose porous matrix which allows unimpaired entry of macromolecules. Some of the derivatives of Sepharose which have been useful for coupling ligands containing either carboxyl or amino groups are the ??aminoalkyl, bromoacetyl and diazonium Sepharoses. Polyacrylamide can be derivatized by conversion to the azide, polystyrenes can be nitrated, reduced and diazotized and nylon can be partially hydrolysed to liberate free carboxyl and amino groups. The necessity of interposing a spacer arm between the ligand and the matrix has also been discussed. Some applications of affinity techniques for the purification of enzymes, antibodies, receptors and cell organelles have been dealt with briefly. This is followed by a brief outline of the use of affinity chromatography for (i) purification of the gene II protein (ii) identification of strychnine?sensitive sites on neurons. Section?II: This section is divided into three chapters. Chapter?I: This chapter briefly reviews the field of filamentous bacterial viruses, with special reference to M13. The mode of adsorption, penetration, replication and release of phage—in short, all events in the life cycle of the phage—have been discussed. The filamentous phages have single?stranded DNA and are distinct from other phages in being neither lytic nor lysogenic. The phage particles ‘ooze’ out through pores in the membrane. Eight genes and gene?protein correlations have been made for M13, and the role of each of these has been discussed. The product of gene II has been implicated in diverse functions in the life cycle of M13: (i) replication of RF (ii) changes in surface properties of infected cells (iii) release of lipopolysaccharide (LPS). LPS is released from cells infected with wild?type M13, but not from those infected with am2 mutants. The loss of LPS is probably responsible for changes in the surface properties. Since LPS is a component of the membrane of the host cell, the site where RF replication also occurs in M13, the M13 gene II protein may specifically bind with both LPS and DNA. Chapter?II: This chapter describes the preparation of two types of affinity systems for the chromatography of the gene II protein. The first system was one wherein LPS was covalently linked to a polystyrene backbone through a spacer arm. The second was a DNA?Sepharose matrix since the gene II protein was also expected to have some affinity for DNA. The latter system was more successful for the purification of the protein, though a further purification on Sephadex G?100 was necessary to give the homogeneous protein. This chapter also describes two endonuclease assays for the isolated protein, using ³²P?labelled M13 phage DNA as a substrate. Chapter?III: This chapter discusses the results obtained using the LPS and DNA affinity columns. The gene II protein was more specifically bound on the DNA column and showed a feeble but positive endonuclease activity.

Section?III: This section is also divided into three chapters. Chapter?I: This chapter commences with a discussion on the criteria for identification, localisation and characterization of central neurotransmitters, with special reference to the inhibitory transmitters glycine and gamma?aminobutyric acid. The physiological, biochemical, histochemical and other techniques which have been crucial for developments in this field have been discussed. Compelling evidence indicates that glycine is an inhibitory transmitter in the spinal cord: (i) It is present in pre?synaptic neurons of the spinal cord. (ii) It simulates the hyperpolarisation produced in the post?synaptic membrane by the authentic inhibitory transmitter. (iii) It is specifically antagonised by the alkaloid strychnine. Evidence is available that strychnine inhibits glycine action by binding at or near the site of the glycine receptor on the post?synaptic membrane. If one can locate the strychnine?binding sites, further studies would reveal whether these areas also bind glycine. The mapping of the strychnine?binding sites has been possible by using a specific antibody to strychnine. The identification of several neurotransmitter receptors has also been briefly discussed. Chapter?II: This chapter begins with a description of the preparation of a strychnine?BSA conjugate. Strychnine was converted to the nitro and then to the amino strychnine. The amino strychnine was diazotized and coupled to BSA. Procedures for immunization and collection of sera are also indicated. Strychnine?specific antibodies were purified on strychnine?succinyl aminoethyl Sepharose columns. The fluorescent?labelled antibody was prepared by coupling the antibody to fluorescein isothiocyanate. Spinal cord sections (1 ?) of a monkey which had been made to convulse with excess strychnine were checked for specific binding of the fluorescent?labelled antibody by fluorescence microscopy. Synaptosomal fractions, prepared by standard procedures from strychninised cord, were also examined for binding of the fluorescent?labelled antibody by fluorimetric methods. Electron microscopy was used for identifying the subcellular fractions. Chapter?III: In this chapter the results obtained by fluorescence microscopy and fluorimetry are discussed. Specific binding of the labelled antibody could be demonstrated in both instances for strychninised samples as compared to the controls.