Nicotine synthesis was stimulated by reduction of the medium auxin concentration (induction medium) in callus tissue originating from Nicotiana tabacum cv. Samsun. The enzyme activities of the route ornithine to methylpyrroline, which are those of ornithine decarboxylase, putrescine methyltransferase and methylputrescine oxidase, were determined during callus growth in the induction medium and as a control under non-nicotine-stimulating conditions (growth medium). The enzymes were assayed by high-performance liquid chromatography. Whereas the activities of ornithine decarboxylase were very similar under nicotine-stimulating and non-stimulating conditions, those of putrescine methyltransferase and methyl-putrescine oxidase increased strongly in the induction medium. In addition, the pools of putrescine and methylputrescine were determined throughout the callus growth cycle. Both sets of data strongly confirm the supposition that putrescine methyl-transferase is the enzyme under stringent control for nicotine biosynthesis, whereas the subsequent methylputrescine oxidase is co-regulated, although less stringently.
In tobacco callus, the induction of nicotine synthesis, which stimulates enzyme activities of the ornithine-methylpyrroline route (see the preceding paper), also leads to marked changes in the enzyme activities of the pyridine-nucleotide cycle. This cycle provides the metabolite (probably nicotinic acid) for condensation with methylpyrroline to produce nicotine. The activities of eight enzymes of the pyridine-nucleotide cycle and of quinolinic-acid phosphoribosyltransferase, the anaplerotic enzyme, were determined by high-performance liquid chromatography assays. The distinct changes of their activities upon induction of nicotine synthesis lead to the following conclusions: i) nicotinic acid is the relevant metabolite which is provided by the pyridine-nucleotide cycle and consumed for nicotine synthesis. ii) The enhancement of the nicotinic-acid pool arises in two ways, by synthesis of NAD and degradation via nicotinamide mononucleotide and by a direct route from nicotinic-acid mononucleotide (NaMN) which is degraded by a glycohydrolase with a rather high K m value. Such a K m value prevents the complete depletion of the NaMN pool.
Neuropeptide Y (NPY) content, NPY receptors, and alpha-subunits of the G proteins Go and Gi were determined in cerebral cortex of male normotensive Wistar-Kyoto and spontaneously hypertensive rats at 3-28 wk of age and of adult female rats. NPY lacked major effects on adenylate cyclase or inositol phosphate formation. NPY content was similar in all normotensive groups but lower in spontaneously hypertensive rats at all ages. 125I-NPY labeled a homogeneous population of Y1-like receptors. The Y1 NPY receptor number gradually increased with age with similar values in both strains but was significantly smaller in female than in male rats. The Y1 NPY receptor affinity was similar in all male groups but greater in female rats. The abundance of immunodetectable Go alpha and Gi alpha and of pertussis toxin substrates was less at 3 wk than in older rats but similar in both sexes and strains. We conclude that rat cerebral cortex contains Y1-like receptors; sex, age, and blood pressure differentially regulate NPY content, Y1 NPY receptors, and Go alpha and Gi alpha.
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