New aspects of biosynthesis and metabolism of <Emphasis Type="Italic">N</Emphasis>-acyldopamines in rat tissues

Possible biosynthetic pathways of N-acyldopamines in rat tissues were compared. It was shown that an insignificant amount of the conjugation products was formed during the incubation of arachidonic acid and dopamine, whereas the substitution of tyrosine for dopamine resulted in the productive biosynthesis of N-arachidonoyldopamine. The biosynthesis presumably involves several closely conjugated enzymatic stages, and free fatty acids rather than their CoA esters served as the starting substrates. The decarboxylation stage probably precedes the stage of catechol system formation, because N-acetyltyramine (a probable intermediate) was easily oxidized by monophenol monooxygenase to N-acyldopamine, whereas N-acyltyrosine is hydrolyzed under these conditions. Biosynthesis of N-acyldopamines in a cell-free medium was accompanied by their methylation. The possibility of oxidative metabolism of N-acyldopamines, which could serve as co-substrates or inhibitors of different oxidoreductases, was shown for the first time.     

New aspects of rubber biosynthesis

New aspects of rubber biosynthesis. Following a review of the site of rubber biosynthesis in Hevea brasiliensis and Parthenium argentalum, evidence is given for the initiation of polyisoprene molecules from (ranMerpenoid precursors including geranylgeranyl pyrophosphate. All franj-¹⁴C-geranylgeraniol has been isolated from incubations of H. brasiliensis latex serum with ¹⁴C-isopentenyl pyrophosphate. Gel-filtration chromatography of the serum yields very small rubber particles of high biosynthetic activity, and two proteinaceous fractions. One of these increases the biosynthesis of rubber and may contain the enzyme, isopentenyldiphosphate δ-isomerase, whilst the other appears to inhibit rubber formation. The nature and molecular weight of the rubber formed in vitro is discussed and a mechanism for the de novo formation of rubber particles is suggested.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable sat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable rat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.     

Transaminative pathway of cysteine metabolism in rat tissues.

Enzyme activities of the transaminative pathway of cysteine metabolism in various rat tissues were examined. Liver was found the most active tissue, followed by kidney and heart. Liver and kidney were more pronounced in mercaptopyruvate sulfurtransferase activity than in cysteine transaminase activity; heart was more active in the latter. Red blood cells, which have pronounced sulfurtransferase activity, exhibited no transaminase activity, indicating the pathway is negligible in this tissue.     

Leukotriene A4: metabolism in different rat tissues

The transformation of leukotriene A4 into dihydroxyeicosatetraenoic acids and sulfidopeptide leukotrienes was determined in homogenates of rat tissues supplied with glutathione and albumin. The highest production of leukotriene B4 was found in spleen, lung and small intestine, while leukotriene C4 dominated in liver and lung. 5(S),6(R)-Dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5,6-DHETE) was formed in all tissues, most prominently in kidney, heart and brain. We also found another isomer of 5,6-dihydroxyeicosatetraenoic acid produced in the kidney. This compound was derived from 5,6-DHETE by isomerization, probably of the 11-cis double bond to 11-trans, and the process appeared to be catalyzed by a membrane-bound factor.     

The metabolism of inositol 4-monophosphate in rat mammalian tissues

Rat brain soluble fraction contains an enzymatic activity that dephosphorylates inositol 1,4-bisphosphate (Ins(1,4)P2). We have used anion exchange h.p.l.c. in order to identify the inositol monophosphate product of Ins(1,4)P2 hydrolysis (i.e. Ins(1)P1, Ins(4)P1 or both). When [3H]Ins(1,4)P2 was used as substrate, we obtained an inositol monophosphate isomer that was separated from the co-injected standard [3H]Ins(1)P1. This suggested an Ins(1,4)P21-phosphatase pathway leading to the production of the inositol 4-monophosphate isomer. The dephosphorylation of [32P]Ins(4)P1 was measured in rat brain, liver and heart soluble fraction and was Li+-sensitive. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved a monophosphate phosphatase activity that hydrolyzed both [3H]Ins(1)P1 and [4-32P]Ins(4)P1 isomers.     

Phytosterol feeding induces alteration in testosterone metabolism in rat tissues

The objective of the present study was to examine the metabolism of testosterone in rat tissues as influenced by dietary phytosterols. Testosterone metabolism includes reductions to more active metabolites or aromatization to estrogen. Both higher levels of androgens and estrogens are implicated as risk factors in the development of prostate cancer. Tissues studied included liver, testis, and prostate. Feeding 2% phytosterols with 0.2% cholic acid to rats for 22 days resulted in a 33% reduction in serum testosterone compared with controls, which received only 0.2% cholic acid in the diet. 5-α-Reductase was reduced by 41 to 44% and 33% in the liver and prostate, respectively. No effect of phytosterols was observed in the testis. Only aromatase activity of the prostate was reduced by 55% upon feeding phytosterols. It was concluded that dietary phytosterols may reduce the risk of prostate cancer by lowering the activities of the enzymes of testosterone metabolism.     

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.     

Quantitative properties and biosynthesis of phospholipids in rat embryonic tissues in the process of development

The content of lipid phosphorus and the rate of [1-14C]palmitate incorporation into individual phospholipids of rat embryonic liver, kidney, spleen, brain, and placenta at different stages of prenatal development were studied. It was shown that the level of neutral phospholipids at all stages is much higher than that in acid fractions; however, the rate of the acid fraction exchange is 10 times higher depending on the age of the embryos. The specific radioactivity of individual fractions of embryonic rat tissue largely exceeds that in adult animals.     

Ethylene biosynthesis in fruit tissues

Tracer studies with avocado tissues indicate that methionine is converted to ethylene at stages of the climacteric rise and the climacteric peak, but not at the preclimacteric stage. The results suggest that the control of ethylene biosynthesis is at a step after methionine is synthesized. The endogenous content of methionine was found to be so low that methionine must be actively turned over for ethylene biosynthesis during the stages when the rate of ethylene production is high. Oxygen was found to be essential for this conversion, indicating that at least one of the steps in conversion of methionine to ethylene is oxygen-dependent. The ability of methionine and its keto analogue (α-keto-γ-methylthiobutyric acid) to serve as ethylene precursors by apple tissues was compared. Chemical and kinetic evidence support the view that methionine is a closer precursor of ethylene than its keto analogue.     

D-Amino acid metabolism in mammals: biosynthesis, degradation and analytical aspects of the metabolic study

It was believed for long time that d-amino acids are not present in mammals. However, current technological advances and improvements in analytical instruments have enabled studies that now indicate that significant amounts of D-amino acids are present in mammals. The most abundant D-amino acids are D-serine and D-aspartate. D-Serine, which is synthesized by serine racemase and is degraded by D-amino-acid oxidase, is present in the brain and modulates neurotransmission. D-Aspartate, which is synthesized by aspartate racemase and degraded by D-aspartate oxidase, is present in the neuroendocrine and endocrine tissues and testis. It regulates the synthesis and secretion of hormones and spermatogenesis. D-Serine and D-aspartate bind to the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and function as a coagonist and agonist, respectively. The enzymes that are involved in the synthesis and degradation of these D-amino acids are associated with neural diseases where the NMDA receptors are involved. Knockout mice for serine racemase and D-aspartate oxidase have been generated, and natural mutations in the d-amino-acid oxidase gene are present in mice and rats. These mutant animals display altered behaviors caused by enhanced or decreased NMDA receptor activity. In this article, we review currently available studies on D-amino acid metabolism in mammals and discuss analytical methods used to assay activity of amino acid racemases and D-amino-acid oxidases.     

Quantitative aspects of free fatty acid metabolism in the fasted rat

Palmitate-1-(14)C was injected intravenously into unanesthetized, fasted rats. Disappearance of tracer from plasma free fatty acids was studied. A large component of free fatty acid (FFA) recycling was directly demonstrated by reinjection experiments. The latter studies also indicated the existence of an unidentified, rapidly turning over polar lipid in plasma which was synthesized from palmitate-(14)C. The appearance of (14)C in hepatic and extrahepatic triglycerides, in other esters, and in respired CO(2) was also followed. The data were analyzed using a multicompartmental model and a digital computer. Only a small fraction of the triglycerides formed in liver was derived directly from plasma free fatty acids. The major portion of net triglyceride formation appeared to be by way of an intermediate nontriglyceride ester pool which turned over relatively slowly compared to plasma free fatty acids. Initial approximations are as follows ( micromoles of fatty acid per min per 100 g body weight): net free fatty acid mobilization (irreversible disposal) = 2.4; hepatic triglyceride formation directly from plasma free fatty acid = 0.1; total hepatic lipid formation from plasma free fatty acids = 0.5; oxidation of free fatty acids to CO(2) = 0.8; percentage of respired CO(2) from direct oxidation of fatty acids = 12%; extrahepatic triglyceride formation directly from fatty acids = 0.4; total extrahepatic lipid formed directly from fatty acids = 1.2.