Phenolic metabolism in petunia tissues. I. Characteristic responses of enzymes involved in different steps of polyphenol synthesis to different hormonal influences.

Pronounced changes in enzymatic patterns occur in petunia tissues when calluses are subcultured on media containing different growth substances. As judged by variations of enzymes related to primary metabolism (6-phosphogluconate and malate dehydrogenases) there are individual responses for each metabolic pathway. Concerning the enzymes of aromatic metabolism: (a) Phenylalanine ammonia-lyase, cinnamate and p-coumarate hydroxylases and the enzyme(s) activating phenylpropanoid units vary in the same manner. (b) Chalcone-flavanone isomerase, a key enzyme in the synthesis of flavonoids, and coniferyl alcohol dehydrogenase, which leads to the monomers of lignins, have, on the other hand, an independent behaviour. These responses show that the enzymes involved in the synthesis and activation of phenylpropanoid units seem to act coordinately in plants. Moreover, the data suggest that the common pathway leading to the activated cinnamic acids and the specific metabolic steps of lignin and flavonoid synthesis are regulated in a different way.     

In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues.

A powerful technique is described to localize the activities of a range of enzymes in a wide variety of plant tissues. The method is based on the coupling of the enzymatic reaction to the reduction of NAD and subsequent reduction and precipitation of nitroblue tetrazolium. Enzymes that did not reduce NAD could be visualized by coupling their activities to glucose-6-phosphate dehydrogenase activity via one or more intermediary 'coupling' enzymes. The method is shown to be applicable for the detection of the activities of hexokinase, fructokinase, sucrose synthase, uridine 5'-diphospho-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase, phosphoglucomutase, and phosphoglucose isomerase. It could be used for all tissues tested, including green leaves, stems, roots, fruits, and seeds. The method is specific, very sensitive, and has a high spatial resolution, giving information at the cellular and the subcellular level. The localization of sucrose synthase, invertase, and uridine 5'-diphospho-glucose pyrophosphorylase in transgenic potato plants, carrying a cytokinin biosynthesis gene, is studied and compared with wild-type plants.     

Constitutive synthesis of enzymes involved in 2-aminophenol metabolism and inducible synthesis of enzymes involved in benzoate, p-hydroxybenzoate, and protocatechuate metabolism in Pseudomonas sp. strain AP-3

Pseudomonas sp. strain AP-3 grows on benzoate, p-hydroxybenzoate, protocatechuate, and 2-aminophenol as sole carbon and energy source. This strain converted benzoate and p-hydroxybenzoate to catechol and protocatechuate respectively, which were metabolized via the ortho-cleavage pathway. The enzymes responsible for these reactions were shown to be inducible. In contrast, strain AP-3 constitutively expresses the enzymes involved in the metabolism of 2-aminophenol.     

Expression of enzymes involved in estrogen metabolism in human prostate.

There is evidence that estrogens can directly modulate human prostate cell activity. It has also been shown that cultured human prostate cancer LNCaP can synthesize the active estrogen estradiol (E2). To elucidate the metabolism of estrogens in the human prostate, we have studied the expression of enzymes involved in the formation and inactivation of estrogens at the cellular level. 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2, 4, 7, and 12, as well as aromatase mRNA and protein expressions, were studied in benign prostatic hyperplasia (BPH) specimens using in situ hybridization and immunohistochemistry. For 17beta-HSD type 4, only in situ hybridization studies were performed. Identical results were obtained with in situ hybridization and immunohistochemistry. All the enzymes studied were shown to be expressed in both epithelial and stromal cells, with the exception of 17beta-HSD types 4 and 7, which were detected only in the epithelial cells. On the basis of our previous results, showing that 3beta-HSD and 17beta-HSD type 5 are expressed in human prostate, and of the present data, it can be concluded that the human prostate expresses all the enzymes involved in the conversion of circulating dehydroepiandrosterone (DHEA) to E2. The local biosynthesis of E2 might be involved in the development and/or progression of prostate pathology such as BPH and prostate cancer through modulation of estrogen receptors, which are also expressed in epithelial and stromal cells.     

Adenosine triphosphate hydrolysis in rat dental tissues. A histochemical study to differentiate the enzymes involved.

The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates: beta-glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde. By comparing the enzymatic stainings obtained with the various substrates and at the different pH:s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5'-nucleotidase, inorganic pyrophosphatase, 3':5'-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 min.     

Response of enzymes involved in the metabolism of polyamines to phytohaemagglutinin-induced activation of human lymphocytes.

The stimulation of lymphocyte ornithine decarboxylase and adenosylmethionine decarboxylase produced by phytohaemagglutinin was accompanied by an equally marked, but delayed, stimulation of spermidine synthase, which is not commonly considered as an inducible enzyme. In contrast with the marked stimulation of these biosynthetic enzymes, less marked changes were observed in the biodegradative enzymes of polyamines in response to phytohaemagglutinin. Diamine oxidase activity was undetectable during all stages of the transformation. The activity of polyamine oxidase remained either constant or was slightly decreased several days after addition of the mitogen. The activity of polyamine acetylase (employing all the natural polyamines as substrates) distinctly increased both in the cytosolic and crude nuclear preparations of the cells during later stages of mitogen activation. Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, although powerfully inhibiting ornithine decarboxylase, produced a gradual enhancement of adenosylmethionine decarboxylase activity during lymphocyte activation, without influencing the activities of the two propylamine transferases (spermidine synthase and spermine synthase).     

Energy metabolism reactions in ruminant muscle: responses to age, nutrition and hormonal status

Energy expenditure in muscle comprises reactions related to intermediary metabolism and those of posture and activity. The metabolic reactions respond to a wide range of nutritional and hormonal stimuli and are often apparently co-ordinated; in magnitude, however, their contribution to energy requirements can be minor compared with locomotion and posture. Metabolic reactions include protein turnover, ion transport and substrate cycles. In young ruminants muscle protein synthesis responds to intake but effects on energy expenditure are less pronounced; the situation with the adult is unclear. The involvement of insulin in ruminants may differ from that in monogastrics but effects are observed with thyroid hormones. Ruminant muscle may have a higher energy requirement for Na+, K+ transport which responds in proportion to total oxygen uptake to alterations in intake. Thyroid hormone treatment and, probably, the catecholamines enhance both Na+, K+ and Ca2+ transport. Muscle has fewer substrate cycles than liver and each may contribute only 1-3% toward oxygen consumption. Several are sensitive to insulin, but larger responses are observed with thyroxine and epinephrine and under stress conditions, therefore, may account for significant proportions of heat increment. Energy costs of standing may be considerable and posture movements may change with diet quality and quantity. Locomotory activity may mask changes in the contribution of metabolic reactions in response to different stimuli. Approximately 80% of energy costs for muscle in vivo are accounted for by protein turnover (20-25%), ion transport (25-30%), substrate cycling (5-8%) and standing (30%). Better integration of experiments in vivo and in vitro is required to improve the quantification and resolve data anomalies.     

Effect of prostaglandins in vivo on enzymes involved in thyroid carbohydrate metabolism.

Treatment of adult guinea pigs with prostaglandins produces changes in the levels of enzymes involved in carbohydrate metabolism of the thyroid gland. A decrease in glucose-6-phosphate dehydrogenase activity is observed with a concomitant increase in 6-phosphogluconic dehydrogenase; the glycolytic enzymes appear unaffected by the same treatment. The results indicate that prostaglandins do not have the biochemical effects obtained with thyrotropin and cAMP administration, showing that these compounds play an antagonistic role in comparison with the above mentioned stimulating agents.     

The level of enzymes involved in the allantoin metabolism of Bacillus fastidiosus grown under different conditions

Bacillus fastidiosus was cultivated in batch and continuous culture on various carbon and nitrogen sources. The enzymes involved in allantoin degradation (allantoinase, urease, carboligase) of B. fastidiosus were hardly affected by either carbon or nitrogen source. In contrast, the enzymes involved in glycerol utilization (glycerol kinase, glycerol 3-phosphate dehydrogenase) were induced during growth on glycerol, but were not affected by the amount of allantoin present.     

Differential binding of proteins to peroxisomes in rat hepatoma cells: unique association of enzymes involved in isoprenoid metabolism.

Farnesyl diphosphate synthase (FPPS: EC2.5.1.10), a key enzyme in isoprenoid metabolic pathways, catalyzes the synthesis of farnesyl diphosphate (FPP) an intermediate in the biosynthesis of both sterol and non-sterol isoprenoid end products. The localization of FPPS to peroxisomes has been reported (Krisans, S. K., J. Ericsson, P. A. Edwards, and G. A. Keller. 1994. J. Biol. Chem. 269: 14165;-14169). Using indirect immunofluorescence and immunoelectron microscopic techniques we show here that FPPS is localized predominantly in the peroxisomes of rat hepatoma H35 cells. However, the partial release of 60;-70% of cellular FPPS activity is observed by selective permeabilization of these cells with digitonin. Under these conditions, lactate dehydrogenase, a cytosolic enzyme, is completely released whereas catalase, a known peroxisomal enzyme, is fully retained. Digitonin treatment of H35 cells differentially affects the release of other peroxisomal enzymes involved in isoprenoid metabolism. For instance, mevalonate kinase and phosphomevalonate kinase are almost totally released (95% and 91%, respectively), whereas 3-hydroxy-3-methylglutaryl-CoA reductase is fully retained. Indirect immunoflourescence studies indicate that FPPS is localized in peroxisomes of Chinese hamster ovary (CHO)-K1 cells but is dispersed in the cytosol of ZR-82 cells, a mutant that lacks peroxisomes. Unlike in H35 cells, FPPS is completely released upon digitonin permeabilization of CHO-K1 and ZR-82 cells. In contrast, under the same permeabilization conditions, catalase is fully retained in CHO-K1 cells but completely released from ZR-82 cells. These studies indicate that FPPS and other enzymes in the isoprenoid biosynthetic pathways, involved in the formation of FPP, are differentially associated with peroxisomes and may easily diffuse to the cytosol. Based on these observations, the significance and a possible regulatory model in the formation of isoprenoid end-products are discussed.     

Neonatal sympathectomy compromises development of responses of ornithine decarboxylase to hormonal stimulation in peripheral tissues

The onset of sympathetic innervation has been shown to play a role in the development of postsynaptic reactivity to stimulation. In the current study, we examined whether this relationship extends to responses evoked by hormonal stimuli. Rats denervated at birth by 6-hydroxydopamine treatment showed an impaired ability of vasopressin or angiotensin to stimulate cardiac ornithine decarboxylase activity. In the kidney, responsiveness was affected only for vasopressin and in the lung denervation had only transient effects on the hormonal responses. These results confirm that sympathetic input is required for proper development of some, but not all hormonal responses; the tissue specificity suggests a role of neural factors selective for cardiac development.     

Metabolism of glucose 1,6-P2. I. Enzymes involved in the synthesis of glucose 1,6-P2 in pig tissues

Pig tissues show four enzymatic activities of glucose 1,6-P2 synthesis: (A) 2 [glucose 1-P]----glucose 1,6-P2 + glucose; (B) glucose 1-P + ATP----glucose 1,6-P2 + ADP; (C) glucose 1-P + fructose 1,6-P2----glucose 1,6-P2 + fructose 6-P; (D) glucose 1-P + glycerate 1,3-P2----glucose 1,6-P2 + glycerate 3-P. Brain is the tissue with highest capability of glucose 1,6-P2 synthesis. With the exception of skeletal muscle, activity "D" represents the highest activity of glucose 1,6-P2 synthesis. In muscle, activity "B" is the major activity. The existence of a specific glucose 1,6-P2 synthase which catalyzes reaction "D" is confirmed. Two peaks of such an enzyme are isolated by ion-exchange chromatography. There is an enzyme which specifically catalyzes reaction "C", not previously described. There is a glucose 1-P kinase not identical to phosphofructokinase.     

Sink metabolism in tomato fruit : I. Developmental changes in carbohydrate metabolizing enzymes

In developing tomato (Lycopersicon esculentum Mill.) fruit, starch levels reach a peak early in development with soluble sugars (hexoses) gradually increasing in concert with starch degradation. To determine the enzymic basis of this transient partitioning of carbon to starch, the activities of key carbohydrate-metabolizing enzymes were investigated in extracts from developing fruits of three varieties (cv VF145-7879, cv LA1563, and cv UC82B), differing in final soluble sugar accumulation. Of the enzymes analyzed, ADPglucose pyrophosphorylase and sucrose synthase levels were temporally correlated with the transient accumulation of starch, having highest activities in cv LA1563, the high soluble sugar accumulator. Of the starch-degrading enzymes, phosphorylase levels were fivefold higher than those of amylase, and these activities did not increase during the period of starch degradation. Fiften percent of the amylase activity and 45 to 60% of the phosphorylase activity was localized in the chloroplast in cv VF145-7879. These results suggest that starch degradation in tomato fruit is predominantly phosphorolytic. The results suggest that starch biosynthetic capacity, as determined by levels of ADPglucose pyrophosphorylase rather than starch degradative capacity, regulate the transient accumulation of starch that occurs early in tomato fruit development. The results also suggest that ADPglucose pyrophosphorylase and sucrose synthase levels correlated positively with soluble sugar accumulation in the three varieties examined.