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PubMed1995

Use of 4-fluoro-L-ornithine to monitor metabolic flux through the polyamine biosynthetic pathway.

The mechanistic effectiveness of various polyamine analogs and enzyme inhibitors is typically determined by their ability to deplete intracellular polyamine pools. In this study, we describe an assay that may prove useful in augmenting this relatively static assessment of drug action. The assay relies upon the substitution of 4-fluoro-L-ornithine (Fl-Orn) for ornithine as a polyamine precursor to provide a means to measure metabolic flux through polyamine pools. At concentrations up to 500 microM, the analog did not inhibit the growth of L1210 murine leukemia cells during incubations of up to 72 hr. Using HPLC, the analog was processed metabolically over time to what was deduced to be 2-fluoroputrescine, 6-fluorospermidine and 6-fluorospermine. The relative proportion of fluorinated polyamine analog to the natural polyamine increased with time and Fl-Orn concentration. The sum of the two was found to be nearly identical to the respective polyamine pool of control cells exposed instead to 500 microM ornithine. This indicates that Fl-Orn was recognized and utilized as a precursor at a rate very similar to that of ornithine itself. Using L1210 cells at different stages of cell growth, it was determined that the metabolic flux through the pools, as indicated by the rate of appearance of individual fluorinated polyamine species, reflected the proliferation status of the cells--non-growing cells failed to incorporate the analog. Likewise, in cell types with varying polyamine pool profiles, such as polyamine enzyme overproducers or those with constitutively different spermidine of spermine ratios, the incorporation of the fluorinated analogs into pools was found to be proportional to the size to the natural polyamine pool. In cells treated with inhibitors of S-adenosylmethionine decarboxylase, Fl-Orn incorporation indicated a total blockade of polyamine synthesis at that enzyme site. Overall, the Fl-Orn assay has demonstrated that polyamine pool profiles generally reflect the rate of flux through the pathway in proliferating cells, suggesting that most intracellular polyamines are freely exchangeable with those undergoing metabolic flux.

Adenosylmethionine Decarboxylase↗
PubMed1982

A new synaptosomal biosynthetic pathway of proline from ornithine and its negative feedback inhibition by proline.

The biosynthesis of L-proline (Pro) was studied in sonicates of mouse brain synaptosomes (P2). Incubation with L-[14C]glutamic acid (Glu) resulted in formation of [14C]gamma-aminobutyric acid (GABA) and L-[14C]aspartic acid (Asp), but not [14C]Pro. On the other hand, L-ornithine (Orn) was found to be an effective precursor of Pro. Incubation of [3H]Orn with the sonicated mouse brain P2 fraction not only resulted in the extensive formation of [3H]Pro, but also yielded [3H]Glu and [3H]GABA. Almost 50% of [3H]Orn consumed was converted into [3H]Pro. In vitro addition of Pro (0.1-1 mM) inhibited the formation of [3H]Pro from [3H]Orn to the extent of 40-75%, while increasing the accumulations of [3H]Glu and [3H]GABA without affecting the consumption of [3H]Orn. These results clearly demonstrate that a potential site exists for negative feedback inhibition in Pro biosynthesis from Orn in the mammalian central nervous system.

Animals↗
PubMed1985

Effects of lead on the heme biosynthetic pathway in rat kidney.

Exposure of rats to lead in drinking water at concentrations of 500 ppm and 1000 ppm for 3 and 6 months resulted in elevated blood lead levels, formation of kidney intranuclear inclusion bodies, and increased urinary excretion of uroporphyrin and coproporphyrin. The erythrocytic Zn-protoporphyrin was increased in the highest dose group. No significant effects on body weight gain or kidney weight were observed. Renal activity of delta-aminolevulinic acid synthetase (ALAS) was not significantly affected by lead treatment. The renal activity of delta-aminolevulinic acid dehydrase (ALAD) was moderately increased and ferrochelatase activity was significantly decreased. The relatively small effects of chronic lead exposure on renal heme biosynthesis suggests that intracellular complexation of lead with high-affinity renal lead binding proteins (PbBP) and formation of intranuclear inclusions in proximal tubule cells protects this highly sensitive pathway in kidney from lead inhibition in vivo. These data also suggest that the observed increases in urinary porphyrin excretion are primarily due to lead effects on the erythropoietic system.

5-Aminolevulinate Synthetase↗
PubMed1988

Inhibition of cholesterol side-chain cleavage by intermediates of an alternative steroid biosynthetic pathway.

Mitochondrial preparations from endocrine tissues were incubated with radioactive cholesterol and the effect of hydroxylated metabolites of 23,24-dinor-5-cholen-3 beta-ol (23,24-dinor-5-cholene-3 beta,20-diol and 23,24-dinor-5-cholene-3 beta,21-diol) on the production of pregnenolone was measured. These compounds are intermediates in an alternative, sesterterpene pathway for steroid hormone biosynthesis. It was found that these materials, like the analogous side-chain-hydroxylated derivatives of cholesterol (20 alpha-hydroxycholesterol and 22S-hydroxycholesterol), inhibit cholesterol side-chain cleavage. The possibility that there could be a control mechanism whereby metabolites of 23,24-dinor-5-cholen-3 beta-ol inhibit steroidogenesis occurring by the cholesterol pathway is discussed.

Adrenal Glands↗
PubMed1988

The Saccharomyces cerevisiae ARO1 gene. An example of the co-ordinate regulation of five enzymes on a single biosynthetic pathway.

The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae 'general control' mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.

Alcohol Oxidoreductases↗
PubMed1995

Complete separation of the beta,epsilon- and beta,beta-carotenoid biosynthetic pathways by a unique mutation of the lycopene cyclase in the green alga, Scenedesmus obliquus.

The mutant, C-2A'-34, lacks the beta, epsilon-carotenoids, alpha-carotene, lutein and loroxanthin. When grown under heterotrophic or mixotropic conditions this strain develops significantly higher levels of beta-carotene and violaxanthin than does the original developmental mutant of Scenedesmus, C-2A'. The decrease in chlorophyll a and chlorophyll b observed in C-2A'-34 is accompanied by the near absence of the LHC. The light intensity dependence of greening of this strain is comparable to that of C-2A'; the loss of the beta,epsilon-carotenoids and modification of the pool of beta,beta-carotenoids neither prevent the proximal pigment-protein complexes of photosystems I and II from developing nor cause any short term photosensitivity. The increase in the beta,beta-carotenoids in C-2A'-34 apparently compensates for the loss of the beta,epsilon-carotenoids required in formation of the proximal and distal antennae systems but not in the LHCs.

Carotenoids↗
PubMed1989

Biosynthetic pathways of testosterone and estradiol-17 beta in slices of the embryonic ovary and testis of the chicken (Gallus domesticus).

To elucidate synthetic pathways of testosterone and estradiol-17 beta in embryonic gonads of the chicken, metabolism of various 14C-labeled steroids in slices of the left ovaries and paired testes of 15- and 9-day-old chicken embryos was examined. (1) Fifteen-day-old chicken embryos: From pregnenolone, more 17 alpha-hydroxypregnenolone was produced than progesterone in the ovary, while more progesterone was produced than 17 alpha-hydroxypregnenolone in the testis. From 17 alpha-hydroxypregnenolone, however, only dehydroepiandrosterone was detected as a product in both gonads. Dehydroepiandrosterone was converted mainly into androstenedione and its 5 beta-reduced derivatives by both gonads. Progesterone was converted into 5 beta-pregnane-3,20-dione more than into 17 alpha-hydroxyprogesterone by both gonads. Both gonads metabolized 17 alpha-hydroxyprogesterone, androstenedione, and testosterone predominantly into their corresponding 5 beta-reduced steroids, while production of androstenedione from 17 alpha-hydroxyprogesterone and of testosterone from androstenedione was limited. Estradiol-17 beta was produced from androstenedione and testosterone only by the ovary. (2) Nine-day-old chicken embryos: From pregnenolone, production of progesterone and 17 alpha-hydroxypregnenolone was similar in the ovary. On the other hand, in the testis, more progesterone was produced than 17 alpha-hydroxypregnenolone from pregnenolone. For delta 4-3-oxo steroids, strong activity of 5 beta-reductase was demonstrated in both gonads. From these results, both delta 4- and delta 5-pathways are involved in the formation of testosterone and then finally of estradiol-17 beta by the embryonic gonads of the chicken, and relative preference for the pathway seems to depend on sexes and embryonic ages. In addition, it is suggested that steroidogenesis in these embryonic gonads is characterized by marked activity of 5 beta-reductase, irrespective of sexes or ages.

17-alpha-Hydroxypregnenolone↗
PubMed1991

Production and utilization of farnesoic acid in the juvenile hormone biosynthetic pathway by corpora allata of larval Diploptera punctata.

The regulation of juvenile hormone III (JH III) biosynthesis and release by the corpora allata (CA) was studied in penultimate and final instar male and female larvae of Diploptera punctata using an in vitro radiochemical assay. Final instar female CA produced JH III (2 to 6 pmol.hr-1 per pair) only during the first 8 days of the 20-day stadium, but produced and released farnesoic acid (FA), a JH III precursor, (0.5 to 2.0 pmol.hr-1 per pair), during the entire stadium. FA was identified by cochromatography on TLC of both the product from CA incubations with [3H]FA standard as well as by esterification of biosynthesized FA to methyl farnesoate (MF). Biosynthesis of MF and JH III acid occurred at less than 0.25 and less than 2 pmol.hr-1 per pair, respectively. Because regression analysis of the time course of JH III release over 6 hr showed a constant rate of release after a lag period lasting up to 1.7 hr, rates of JH release of untreated CA (spontaneous) and FA-treated CA (stimulated) were determined by 2-hr sequential incubations of CA, following a 2-hr preincubation. FA-treated rates of JH III release were higher than spontaneous rates. In penultimate instars, the decline in the spontaneous rates during the latter half of the stadium was accompanied by a reduced degree of FA stimulation. In final instars, as the spontaneous rates declined between Days 2 and 6, the magnitude of the stimulation of JH III biosynthesis by FA increased, whereas in the second half of the stadium, the degree of stimulation was very low. Addition of exogenous FA to CA from late final instars increased FA production significantly more than JH III production. This apparent stimulation of FA biosynthesis is evident in CA with both high and low spontaneous rates of JH release and suggests that rate limitation of JH III biosynthesis in the final stadium occurs in part at the level of the o-methyl transferase which converts FA into MF.

Animals↗
PubMed1983

The stimulatory effect of corticotropin on cortisol biosynthetic pathway in guinea-pig adrenocortical cells.

The mechanism of the prolonged stimulatory effect of corticotropin (ACTH) on adrenocortical synthesis of cortisol was studied in guinea-pig adrenocortical cells harvested from control animals and from guinea-pigs submitted 24 h before the sacrifice to a prolonged ether anesthesia in an attempt to induce a release of endogenous ACTH. As a result of this in vivo exposure to endogenous ACTH, the maximal capacity to produce glucocorticoids (by 1 X 10(5) cells incubated during 2 h) in response to ACTH increased from 579 +/- 111 ng (control group) to 915 +/- 143 ng for cells from treated animals, whereas the apparent affinity of the steroidogenic response to ACTH remained unchanged. This hyper-reactivity of cells from anesthetized animals was also evident in the presence of dibutyryl cyclic AMP. Moreover, there was increased conversion of exogenous pregnenolone into cortisol by cells from previously anesthetized animals. It was therefore concluded that ACTH increases in a lasting way the activity of steroidogenic pathway leading to cortisol synthesis by adrenocortical cells at sites distal to cyclic AMP generation. Besides an obvious increase of formation of pregnenolone in response to ACTH, it seems that this ACTH-induced enhancement in the capacity of the steroidogenic response to ACTH also implies a prolonged stimulatory influence of the peptide on the post-pregnenolone steroidogenic pathway leading to cortisol synthesis.

Adrenal Cortex↗
PubMed1990

Potential bile acid precursors in plasma--possible indicators of biosynthetic pathways to cholic and chenodeoxycholic acids in man.

The plasma concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid have been compared with that of 7 alpha-hydroxy-4-cholesten-3-one in healthy subjects and in patients with an expected decrease or increase of the bile acid production. In controls and patients with liver disease, the level of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was positively correlated to that of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and not to that of 7 alpha-hydroxy-4-cholesten-3-one. In patients with stimulated bile acid formation the levels of the acids were not correlated to each other but there was a significant positive correlation between the levels of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid and 7 alpha-hydroxy-4-cholesten-3-one. These findings indicate that the precursor of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid differs depending on the activity of cholesterol 7 alpha-hydroxylase. Since the activity of this enzyme is reflected by the level of 7 alpha-hydroxy-4-cholesten-3-one in plasma the findings are compatible with a formation of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid from 3 beta,7 alpha-dihydroxy-5-cholestenoic acid when the rate of bile acid formation is normal or reduced and from 7 alpha-hydroxy-4-cholesten-3-one under conditions of increased bile acid synthesis. In support of this interpretation, 7 alpha,26-dihydroxy-4-cholesten-3-one was identified at elevated levels in plasma from patients with ileal resection or treated with cholestyramine. The levels of 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one were also higher than normal in these patients. Based on these findings and previous knowledge, a model is proposed for the biosynthesis of bile acids in man. Under normal conditions, two major pathways, one "neutral" and one "acidic" or "26-oxygenated", lead to the formation of cholic acid and chenodeoxycholic acid, respectively. These pathways are separately regulated. When the activity of cholesterol 7 alpha-hydroxylase is high, the "neutral" pathway is most important whereas the reverse is true when cholesterol 7 alpha-hydroxylase activity is low. In cases with enhanced activity of cholesterol 7 alpha-hydroxylase, the "neutral" pathway is connected to the "acidic" pathway via 7 alpha,26-dihydroxy-4-cholesten-3-one, whereas a flow from the acidic pathway to cholic acid appears to be of minor importance.

Adult↗