Fractionation of simian virus 40 DNA fragments by RPC-5 column chromatography

This report describes several modifications of the original radioenzymatic assay for serotonin (4) which increase the sensitivity of the assay 10-fold as well as enhance its reliability. Serotonin is converted to [³H]melatonin, in two steps. First, serotonin is acetylated to N-acetylserotonin by acetic anhydride. The N-acetylserotonin is then incubated with hydroxyindole-O-methyltransferase and S-[methyl-³H]adenosyl methionine and is converted to [³H]melatonin. The radioactive melatonin is extracted with toluene-isoamyl alcohol (7:3), dried, reconstituted, isolated by one-dimensional, silica gel, thin-layer chromatography, and counted in a liquid scintillation counter. The assay is specific and sensitive to approximately 5 pg of serotonin and thus can be used to measure serotonin levels in single brain nuclei or microliter quantities of biological fluids. The assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

A SENSITIVE MICROASSAY FOR TRYPTOPHAN HYDROXYLASE IN BRAIN

—A specific and sensitive, radioisotopic microassay for tryptophan hydroxylase (EC 1.14.36) is described, which is capable of determining enzymatic activity in as little as 5 μg of crude brainstem homogenate. 5-Hydroxytryptophan, the immediate product of hydroxylation of tryptophan is enzymatically converted to N-acetylserotonin. A radioisotopic label is then introduced by the enzymatic methylation of N-acetylserotonin in the presence of [³H]methyl-S-adenosyl-methionine. The [³H]-melatonin thus formed is isolated by extraction and counted. With this assay, the activity in individual hypothalamic nuclei (arcuate nucleus, median eminence, suprachiasmatic nucleus, and medial forebrain bundle) has been measured.     

Isotopic microassay of histamine, histidine, histidine decarboxylase and histamine methyltransferase in brain tissue

Abstract— Microassays are described for histamine, histidine, and the activities of the enzymes histidine decarboxylase (EC 4.1.1.22) and histamine niethyltransferase (EC 2.1.1.8) in brain tissue. The enzymic-isotopic microassay for histamine is based on the methylation of tissue histamine by added histamine methyl-transferase and [¹⁴C]- or [³H]-labelled S-adenosyl-l -methionine. In a double-isotopic form of the assay, a tracer of [³H]histamine is employed along with [¹⁴C]S-adenosyl-l -methionine, and the ratio [¹⁴C]:[³H] reflects the amount of histamine in the sample. Because the methylation of histamine is uniform in brain samples studied, a single isotopic assay with [³H]S-adenosyl-l -methionine as the methyl donor is possible and increases sensitivity, so that 10 pg of tissue histamine can be estimated reliably. The assay for histidine involves decarboxylation of histidine by a bacterial histidine decarboxylase and measurement of the histamine formed by the enzymicisotopic procedure. In the histidine decarboxylase assay, histamine synthesized from added histidine is measured. The assay for histamine methyltransferase involves measuring the formation of [¹⁴C]methylhistamine with [¹⁴C]S-adenosyl-l -methionine serving as the methyl donor.     

A radiometric assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A radiometric assay for pyridoxamine 5′-phosphate oxidase (pyridoxamine (pyridoxine) 5′-phosphate:O₂ oxidoreductase (deaminating), EC 1.4.3.5) has been developed utilizing N-(5′-phosphopyridoxyl)[³H]tryptamine. This assay is more sensitive than previously used colorimetric and fluorescent assays for this oxidase and furthermore is applicable to erythrocytes. Tritiated substrate is incubated with an enzyme sample in the presence of excess unlabeled truptamine and the radiolabeled tryptamine product is extracted into toluene and quantitated by liquid scintillation counting.     

A rapid radioenzymatic assay for dopamine and N-acetyldopamine.

Dopamine (DA) was measured in various tissue extracts as [³H]methoxy-N-acetyldopamine after incubation with two partially purified enzymes, catechol-O-methyl transferase (EC 2.1.1.1) and N-acetyltransferase (EC 2.3.1.5), in the presence of [³H]adenosylmethionine and acetyl-CoA. This product can be separated quantitatively from labeled products of norepinephrine and epinephrine by solvent extraction. N-Acetyl-DA can be assayed by omitting the acetylating system from the incubation mixture. The procedure is rapid, convenient for processing large numbers of samples, and has a sensitivity of approximately 0.1 pmol. It has been used to measure DA in ganglia and in individual neurons from gastropod mollusks.     

The Phosphoinositide Signal Transduction System Is Impaired in Bipolar Affective Disorder Brain

Abstract: The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [³H]phosphatidylinositol ([³H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca²⁺ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration-dependently activated by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and by NaF. GTPγS-stimulated [³H]PI hydrolysis was markedly lower (50%) at all tested concentrations (0.3–10 µM GTPγS) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTPγS in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTPγS-stimulated [³H]PI hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans-1-aminocyclopentyl-1,3-dicarboxylic acid, serotonin, and ATP each activated [³H]PI hydrolysis above that obtained with GTPγS alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTPγS alone. Thus, among the three cortical regions examined there was a selective impairment in G protein-stimulated [³H]PI hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder.     

Properties of Particulate and Detergent-Solubilized Phospholipid N-Methyltransferase Activity from Calf Brain

Abstract: Calf brain membranes catalyze the enzymatic transfer of [CH₃-³H]methyl groups from S-adenosyl-l -[CH₃-³H]methionine into endogenous phosphatidyl-N-methylethanolamine (PME), phosphatidyl-N,N-dimethylethanolamine (PDE), and phosphatidylcholine (PC). Phospholipid N-methylation can be stimulated by the addition of exogenous PME or PDE, added in aqueous dispersions with sodium taurocholate. When membranes are incubated in the presence of exogenous PME, [CH₃-³H]PDE represents 86% of the labeled phospholipid products. When exogenous PME is replaced by PDE, 91% of the label is incorporated into PC. Thus, under these in vitro conditions it is possible to assay PME- and PDE-N-methyitransferase activity separately. The calf brain phospholipid N-methyltransferase activity has also been solubilized by treating the membranes ultrasonically in the presence of Triton X-100 and 10 mM monothioglycerol. When the detergent extracts are incubated in the presence of exogenous PME, [CH₃-³H]PDE represents 86% of the enzymatically labeled products. In the presence of exogenous PDE, more than 97% of the label is incorporated into PC. Optimal conditions for the membrane-bound and detergent-solubilized PME- and PDE-N-methyltransferase activity have been established. These conditions have been used as a basis for testing the hypothesis that the conversion of PME to PC is catalyzed by a single enzyme in calf brain. In these studies, PME- and PDE-N-methyltransferase activities have been found to be similar, if not identical, with respect to: (1) extractability with Triton X-100; (2) pH optimum; (3) response to divalent cations; (4) apparent K_m, for S-adenosyl-l -methionine and K_I for S-adenosyl-l -homocysteine, (5) sensitivity to N-ethylmaleimide; and (6) thermal inactivation at 55°. Overall, these results are consistent with the conclusion that in calf brain, PME and PDE are methylated by the same enzyme or by two phospholipid N-methyltransferases having very similar properties.     

CATECHOLAMINES IN FETAL AND NEWBORN RAT BRAIN

The levels of dopamine and norepinephrine were determined in the brains of fetal and newborn rats by means of a sensitive, radiometric-enzymatic assay. Catecholamines were converted to their 3-O-methylated derivatives in the presence of catechol-O-methyl transferase (EC 2.1.1.1) and [³ H-methyl]S-adenosylmethionine; and the [³H]-derivatives were isolated by selective extraction. The assay had a sensitivity for dopamine and norepinephrine of 100 picograms and was linear to at least 30 nanograms of catecholamines. Both amines were present at 15 days of gestation and increased 15-fold in content during the last week of gestation. The regional distribution of these neurotransmitters in the brain of the newborn rat correlated with the distribution of their biosynthetic enzymes. An investigation of the effects of reserpine, pheniprazine, α-methyl-para-tyrosine, diethyldithiocarbamate and l -DOPA on the levels of dopamine and norepinephrine in the brains of the 18-day gestational fetus indicated that the levels of these neurotransmitters are under controls similar to those known to occur in the brain of the adult rat.     

PURIFICATION OF RAT BRAIN CHOLINE ACETYLTRANSFERASE AND AN ESTIMATION OF ITS HALF-LIFE

—Acetyl-CoA:choline-O-acetyltransferase (ChAc, EC 2.3.1.6) was purified from rat cerebral cortex and its half-life determined. The molecular weight of the enzyme under non-denaturing conditions was estimated by gel filtration to be in the range of 60,000–65,000. On SDS acrylamide gels, the purified enzyme migrated as a single band with a molecular weight estimated as 62,000. The turnover rate of ChAc in the mature rat was determined by the double label method, employing l -[1-¹⁴C]leucine and l -[4,5-³H]leucine. Its half-life under steady-state conditions was estimated to be 5.2 days. As a control, tubulin was isolated from the same preparation and its half-life measured. Under these conditions tubulin exhibited a half-life of 3.8 days.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

Evidence for the occurrence of polyamine oxidase in the dicotyledonous plant Medicago sativa L. (alfalfa)

Polyamine oxidase (EC 1.5.3.3) activity has not been detected previously in cells of dicotyledonous plants, although it has been characterized extensively in monocotyledonous plants. Evidence is presented in this report for the occurrence of polyamine oxidase in dialyzed crude extracts of the dicotyledonous plant, Medicago sativa L. (alfalfa). Three enzyme assays were used to quantitate the formation of the three products of the reaction catalyzed by polyamine oxidase. 1-Pyrroline formation was measured colorimetrically as a yellow quinazolinium complex with o-aminobenzaldehyde. Hydrogen peroxide formation was measured spectrophotometrically with a coupled peroxidase assay system by peroxidative oxidation of guaiacol. [³H]1,3-Diaminopropane formation was measured by using [1,8-³H]spermidine as the substrate and separating the radiolabelled reaction product from the substrate by paper electrophoresis. This latter assay provided evidence that a polyamine oxidase of type [EC 1.5.3.3] catalyzed the cleavage reaction between a secondary nitrogen atom and an adjacent carbon of the butyl moiety of spermidine. Significant polyamine oxidase activity was detected in floral tissues, cortex tissues of the root, young leaves, and young germinated seedlings of alfalfa. The occurrence of polyamine oxidase in alfalfa accounts for the formation of the essential substrate, 1,3-diaminopropane, required for the biosynthesis of the uncommon polyamines, norspermidine and norspermine, which we have recently detected in alfalfa.Abbreviations PAO polyamine oxidase - MOPS [3-(N-morpholino)propanesulfonic acid] - MES [2-(N-morpholino)ethanesulfonic acid] - TES [N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid] - BICINE [N,N-bis (2-hydroxyethyl)glycine] - DTC diethyldithiocarbamic acid - R_m the distance of migration of a polyamine relative to putrescine after electrophoresis on paper     

Does high affinity [3H] imipramine binding label serotonin reuptake sites in brain and platelet?

Recent studies have demonstrated the presence of high affinity binding sites for [³H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [³H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [³H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [³H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [³H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [³H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of [³]] imipramine selectively labels serotonin uptake sites in brain and platelet.     

Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal neurons

The accumulation of [³H]leucine- and [³H]fucose-labelled axonal proteins, acetyl-CoA : choline O-acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) was studied proximal to a ligature applied to the hypoglossal nerve of the rabbit at different phases of nerve regeneration. After 1 week of regeneration, the accumulation of rapidly migrating [³H]leucine-labelled proteins, ChAc and AChE was reduced as compared to that of the contralateral nerve. In contrast, the accumulation of [³H]fucose-labelled glycoproteins was markedly increased. After a regeneration period of 4-6 weeks, the accumulation of proteins and glycoproteins in the regenerating nerve was increased whereas the accumulation of ChAc and AChE was almost normal. The results indicate an initial depression of the synthesis and axonal transport of the bulk of rapidly migrating proteins, ChAc and AChE in the chromatolytic hypoglossal neurons whereas the synthesis and transport of rapidly migrating glycoproteins is increased. These initial changes are less pronounced during the subsequent regeneration period.     

Roles for Nitric Oxide as an Intra- and Interneuronal Messenger at NMDA Release-Regulating Receptors: Evidence from Studies of the NMDA-Evoked Release of [3H]Noradrenaline and d-[3H]Aspartate from Rat Hippocampal Slices

Abstract: N-Methyl-d -aspartate (NMDA) receptors regulating the release of [³H]noradrenaline ([³H]NA) and d -[³H]aspartate (d -[³H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg²⁺-free Krebs-Henseleit buffer, the NMDA-evoked release of [³H]NA and d -[³H]Asp was Ca²⁺ dependent and inhibited by the NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [³H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d -[³H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d -[³H]Asp appear to be presynaptic, whereas those for [³H]NA are extrasynaptic in location. l -Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [³H]NA (100%) and d -[³H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 µM)—stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [³H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (N^G-nitro-, N^G-methyl-, and N^G-amino-l -arginine, all 100 µM) attenuated (50–80%) the NMDA-stimulated release of [³H]NA and d -[³H]Asp, as did KN-62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.     

A Simple Radioenzymatic Method to Measure Picogram Amounts of DOPA in Brain and Biological Fluids

A simple radioenzymatic method for the determination of DOPA is described. The method is based on the conversion of DOPA to 3-O-[methyl-³H]DOPA by catechol-O-methyltransferase in the presence of S-adenosyl-[methyl-³H]methionine and purification of the labelled product by Sephadex G10 and Dowex 50 W × 4 ion exchange resin. The method has been applied to the assay of endogenous DOPA in different brain areas and to measuring DOPA accumulation after inhibition of aromatic amino acid DOPA decarboxylase.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

α-DIFLUOROMETHYL DOPA, A NEW ENZYME-ACTIVATED IRREVERSIBLE INHIBITOR OF AROMATIC L-AMINO ACID DECARBOXYLASE

DL-x-Difluoromethyl DOPA (DFMD, RMI 71801), an enzyme-activated irreversible inhibitor of aromatic L-amino acid decarboxylase in vitro, produces a rapid, long-lasting and dose-dependent inhibition of aromatic L-amino acid decarboxylase in peripheral tissues of mice when administered i.p. or orally. Doses of 500 mg/kg i.p. produce only very slight inhibition of the enzyme activity in mouse brain whilst inhibiting the enzyme activity of peripheral tissues by more than 90%. With L-[³H]-DOPA co-administration brain concentrations of L-[³H]DOPA and ³H-catecholamines are increased 3- to 8-fold concomitant with a decrease in the peripheral decarboxylation of L-[³H]DOPA. Under these conditions it is clear that the slight inhibition of enzyme activity in the brain is totally inadequate to inhibit the decarboxylation of L-DOPA in this organ. Similarly, the decarboxylation of exogenously supplied 5-hydroxytryptophan is inhibited peripherally with a consequent increase in brain serotonin concentrations. DFMD is another example of an enzyme-activated irreversible inhibitor which due to its novel and specific mechanism of action, may offer advantages over existing decarboxylase inhibitors.     

Substance P-Induced Reduction in the Initial Accumulation of Cytosolic myo-[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo-[³H]inositol. This effect was rapid, because a reduction of ～15% could be seen already at 30 s, with the maximal effect (～45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo-[³H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP-induced reduct on in cytosolic ravo[³H]inositol accumulation was concentration dependent; the EC₅₀ obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg¹, D-Trp⁷⁹, Leu]SP), a SP antagonist, used at a concentration oif 10⁵ A/, gave a competitive shift of the dose-response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo-[³H]inositol. [L-Pro⁹]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo-H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C-terminal fragments were more potent than shorter ones. SP N-terminal fragments and SP free acid were -without effect. [Pro⁷]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC₅₀ values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw-[³H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP-induced reduction in cytosolic myo-[³H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo-[³H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo-[³H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-¹⁴C,5-³H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V_c/V_o ratios from the expression A/(B-A) where A and B represent the ³H/¹⁴C isotope ratios of doubly labeled RuBP and 3-PGA, and V_c and V_o represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-¹⁴C]glucose and [6-³H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.