Affinity Chromatography on Immobilised Nucleotides

Abstract

The synthesis and characterisation of two IMP analogues, 8-(6-aminohexyl)-ionosine 5'-monophosphate, Ahx' IMP, and inosine 2', 3 ' 0 [1-(6-aminohexyl)-levulinic acid amidel-acetal 5'-monophosphate, ( A~X L V~)~' ,~' I M P , is described. These analogues were attached to CNBr-activated agarose through the terminal amino group of the spacer molecule. The immobilised-IMP analogues displayed specificity for the inosine-nucleotide-dependent enzyme, IMP dehydrogenase (IMP : NAD + oxidoreductase, EC 1.2.1.14) but not for the NAD+-dependent enzymes, L-alanine and id-acetate dehydrogenases. Escherichiu coli IMP dehydrogenase could be eluted biospecifically from immobilised 8-substituted and ribose-substituted IMP adsorbents with IMP, XMP and GMP. Multiple peaks of enzyme activity in the elution profiles were interpreted in terms of aggregation of the enzyme. A protocol for the large-scale purification of E. coli IMP dehydrogenase is proposed. Homogeneous enzyme of specific activity 9.1 units/mg was obtained in 50 overall yield, representing 14 mg pure protein from a 20-1 culture of E. coli. The two IMP analogues were inactive as substrates in the IMP dehydrogenase reaction.

Separate regulation of purA and purB loci of Escherichia coli K-12

“…IMP dehydrogenase (guaB) and GMP synthetase (guaA) activities were determined as described by Lambden and Drabble (14). SAMP synthetase (purA) was assayed as described by Rudolph and Fromm (19), except that the GTP-regenerating system of Clonis and Lowe was used (6). SAMP lyase (purB) was assayed by the method of Gots and Gollub (8) with SAICAR as the substrate.…”

Triazine-dye affinity chromatography

“…Preliminary reports of affinity labelling of enzymes with triazine dyes have been published (Lowe et al, 1980: Clonis & Lowe, 1980. However, since most of the dyes are heterogeneous, affinity-labelling data must be interpreted carefully (Lowe et al, 1980)…”

Οξαλικη Οξειδαση Απο Hordeum Vulgare:κατιουσα Επεξεργασια Με Χρωματο- Γραφια Συγγενειασμοριακη Μελετη Και Εφαρμογη Στη Μετρηση Του Οξαλικου Οξεος

“…, 1980a, Hughes et al, 1982. Σε σταθερό όγκο πηκτής (υποκατεστημένης χρωστικής) που εξισορροπείται με το κατάλληλο ρυθμιστικό διάλυμα προστίθενται ίσες ποσότητες ενζύμου.…”