The de novo biosynthesis of platelet-activating factor in rat brain

Platelet-Activating Factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is present in nervous tissue and its function is still unknown. We have demonstrated that rat brain is able to synthesize PAF from 1-alkyl-2-acetyl-sn-glycerol and CDP-choline by a "DTT-insensitive" phosphocholine transferase. This represents the last step of the de novo pathway which apparently is the only one existing in the brain for PAF biosynthesis. The enzyme has a microsomal localization, requires Mg++ and is inhibited by Ca++ as reported for phosphocholine transferase utilizing long-chain diradylglycerols as substrates. However, other properties of PAF-synthesizing enzyme (sensitivity to DTT and dependence on pH) are different from those of phosphocholine transferase responsible for the synthesis of diacyl and long-chain alkylacyl glycerophosphocholines. These observations indicate that a specific enzyme for PAF biosynthesis might exist in rat brain.     

The final step in the de novo biosynthesis of platelet-activating factor. Properties of a unique CDP-choline:1-alkyl-2-acetyl-sn-glycerol choline-phosphotransferase in microsomes from the renal inner medulla of rats

Final steps in the synthesis of platelet activating factor (PAF) occur via two enzymatic reactions: the acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine by a specific acetyltransferase or the transfer of the phosphocholine base group from CDP-choline to 1-alkyl-2-acetyl-sn-glycerol by a dithiothreitol (DTT)-insensitive cholinephosphotransferase. Our studies demonstrate that rat kidney inner medulla microsomes synthesize PAF primarily via the DTT-insensitive cholinephosphotransferase since the specific activity of this enzyme is greater than 100-fold higher than the acetyltransferase. The two cholinephosphotransferases that catalyze the biosynthesis of phosphatidylcholine and PAF have similar Mg2+ or Mn2+ requirements and are inhibited by Ca2+. Also topographic experiments indicated that both activities are located on the cytoplasmic face of microsomal vesicles. PAF synthesis was slightly stimulated by 10 mM DTT, whereas the enzymatic synthesis of phosphatidylcholine was inhibited greater than 95% under the same conditions. The concept of two separate enzymes for PAF and phosphatidylcholine synthesis is further substantiated by the differences in the two microsomal cholinephosphotransferase activities with respect to pH optima, substrate specificities, and their sensitivities to temperature, deoxycholate, or ethanol. Study of the substrate specificities of the DTT-insensitive cholinephosphotransferase showed that the enzyme prefers a lipid substrate with 16:0 or 18:1 sn-1-alkyl chains. Short chain esters at the sn-2 position (acetate or propionate) are utilized by the DTT-insensitive cholinephosphotransferase, but analogs with acetamide or methoxy substituents at the sn-2 position are not substrates. Also, CDP-choline is the preferred water-soluble substrate when compared to CDP-ethanolamine. Utilization of endogenous neutral lipids as a substrate by the DTT-insensitive cholinephosphotransferase demonstrated that sufficient levels of alkylacetylglycerols are normally present in rat kidney microsomes to permit the synthesis of physiological quantities of PAF. These data suggest the renal DTT-insensitive cholinephosphotransferase could be a potentially important enzyme in the regulation of systemic blood pressure.     

Angiotensin converting enzyme predominates in the inner cortex and medulla of the rat kidney

Regional distribution of angiotensin converting enzyme(ACE) in the rat kidney was studied. The ACE activities in the inner cortex and outer medulla were about 10 and 5 times those in the outer cortex, respectively. The activity in the inner medulla or papilla was much the same as that in the outer cortex. Immunofluorescence was greatest in the proximal tubules in the inner cortex, while the outer medulla and the inner medulla or papilla showed a weak fluorescence. The brush border membranes isolated from the inner cortex also possessed about 10 times the ACE activity seen in the outer cortex. The results indicate that the major source of renal ACE is not the proximal convoluted tubules in the outer cortex, but rather the brush border membranes of proximal tubules in the inner cortex. The contribution of ACE in the inner cortex would therefore be predominant.     

Stimulation of the de novo pathway for the biosynthesis of platelet-activating factor (PAF) via cytidylyltransferase activation in cells with minimal endogenous PAF production

Treatment of Ehrlich ascites cells with 2 mM oleic acid causes a greater than 10-fold increase in the formation of platelet-activating factor (PAF; 1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) from the de novo precursor of PAF, 1-[3H]alkyl-2-acetyl-sn-glycerol. Under these conditions, CTP:phosphocholine cytidylyltransferase activity, which is known to catalyze the rate-limiting step in phosphatidylcholine biosynthesis, was stimulated 32% (p less than 0.001) over control cells. Surprisingly, the dithiothreitol-insensitive choline-phosphotransferase activity, which catalyzes the final step in PAF biosynthesis, was reduced approximately 95% in membranes isolated from cells that were pre-treated with 2 mM oleic acid. However, calculations of product formation at this reduced cholinephosphotransferase activity revealed that it was still sufficient to accommodate the increased synthesis of PAF observed in the intact oleic acid-treated cells. Kinetic studies and experiments done with cells treated with phenylmethylsulfonyl fluoride (an acetylhydrolase inhibitor) indicate the various metabolic products formed are derived through the following sequence of reactions: 1-alkyl-2-acetyl-sn-glycerol----1-alkyl-2-acetyl-sn-glycero-3- phosphocholine----1-alkyl-2-lyso-sn-glycero-3-phosphocholine----1-alkyl- 2(long-chain) acyl-sn-glycero-3-phosphocholine. These results indicate PAF is the source of alkylacylglycerophosphocholine through the action of an acetylhydrolase and a transacylase as shown in other cell systems. The relative amounts of PAF, lyso-PAF, and alkylacylglycerophosphocholine produced after treatment of the cells with oleic acid in the absence of the phenylmethylsulfonyl fluoride inhibitor indicate that the acylation rate for lyso-PAF is considerably slower (i.e. rate-limiting) than the deacetylation of PAF by acetylhydrolase. We further conclude that the final step in the de novo pathway for PAF biosynthesis is under the direct control of CTP:phosphocholine cytidylyltransferase, which emphasizes the importance of this regulatory (rate-limiting) step in the biosynthesis of both phosphatidylcholine and PAF.     

A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.     

Renal metabolism and urinary excretion of platelet-activating factor in the rat

The origin of platelet-activating factor (PAF) in the urine remains ill defined. The present study documents that [3H]PAF (3.5 mu Ci) injected into the renal artery of isolated control rat kidney preparations perfused at constant pressure with a cell-free medium containing 1% bovine serum albumin (BSA) was excreted in negligible amounts (0.034%) in the urine, whereas 6% was retained by the kidney. When kidneys were perfused with a BSA-free medium, 0.029 and 71% of the total radioactivity added to the perfusate was recovered in the urine and in the renal tissue, respectively. [3H]PAF urine excretion in proteinuric kidneys from adriamycin-treated rats was still negligible (0.015%). Analysis of the renal tissue-retained radioactivity in control and proteinuric kidneys perfused with 1% BSA indicated metabolism into long chain acyl-sn-glycero-3-phosphorylcholine species, lyso-PAF, glycerols, and intact PAF. Thin layer chromatography analysis of [3H]glycerol fraction in these renal extracts showed two major components comigrating with 1-O-alkylglycerol and 1-O-alkyl-2-fatty acylglycerol. Isolated proximal tubules, but not glomeruli from nephrotic rats exposed to increasing concentrations of BSA (0-4%), had a higher PAF uptake than control tubules for BSA concentrations ranging from 0 to 0.1%. Our findings in the isolated perfused kidneys indicate that, in normal conditions, circulating PAF is excreted in the urine in negligible amounts and that the altered glomerular permeability to proteins does not affect this excretion rate. Moreover, analysis of renal tissue radioactivity documented that the renal metabolism of PAF is comparable in control and nephrotic kidneys.     

Activation of the de novo pathway for pyridine nucleotide biosynthesis prior to ricinine biosynthesis in castor beans

The ricinine content of etiolated seedlings of Ricinus communis increased nearly 12-fold over a 4-day period. In plants quinolinic acid is an intermediate in the de novo pathway for the synthesis of pyridine nucleotides. The only known enzyme in the de novo pathway for pyridine nucleotide biosynthesis, quinolinic acid phosphoribosyltransferase, increased 6-fold in activity over a 4-day period which preceded the onset of ricinine biosynthesis by 1 day. The activity of the remainder of the pyridine nucleotide cycle enzymes in the seedlings, as monitored by the specific activity of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase, was similar to that found in the mature green plant. In the roots of Nicotiana rustica, where the pyridine alkaloid nicotine is synthesized, the level of quinolinic acid phosphoribosyltransferase was 38-fold higher than the level of nicotinic acid phosphoribosyltransferase, whereas in most other plants examined, the specific activity of quinolinic acid phosphoribosyltransferase was similar to the level of activity of enzymes in the pyridine nucleotide cycle itself. A positive correlation therefore exists between the specific activity of a de novo pathway enzyme catalyzing pyridine nucleotide biosynthesis in Ricinus communis and Nicotiana rustica and the biosynthesis of ricinine and nicotine, respectively.     

Increased de novo phospholipid biosynthesis in the stimulated rat exocrine pancreas

The $\text{hisU1820}$ mutant (TA799) of $\text{Salmonella}\text{,}\text{typhimurium}$ shows a substantial increase in the levels of ppGpp (MSI) and of pppGpp (MSII) during several types of metabolic shifts. Noticeable amounts of ppGp (MSIII) are also present post-carbon/energy source downshifts and temperature up-shifts. The increased levels of these guanosine polyphosphates were observed despite the absence of the expected reduction in RNA synthesis upon a nutritional downshift. We, therefore, suggest that the $\text{hisU}$ mutation causes an increase in the accumulation of MSI and MSII; and that ppGpp alone is not sufficient to promote restriction of RNA synthesis during a nutritional transition.     

Formation of 1-alkyl-2-acetyl-sn-glycerols via the de novo biosynthetic pathway for platelet-activating factor. Characterization of 1-alkyl-2-acetyl-sn-glycero-3-phosphate phosphohydrolase in rat spleens

1-Alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) is an important intermediate in the biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) from 1-alkyl-2-lyso-sn-glycero-3-phosphate (alkyllyso-GP) via the de novo pathway. In the present investigation, we have characterized a 1-alkyl-2-acetyl-sn-glycero-3-phosphate (alkylacetyl-GP) phosphohydrolase in rat spleens that catalyzes the conversion of alkylacetyl-GP to alkylacetyl-G. The bulk of the enzymatic activity (53%) is located in the microsomal fraction, whereas 28% of the activity is present in mitochondria. The microsomal enzyme has an optimal pH of 7.0-7.4, an "apparent" Km of 31.8 microM for alkylacetyl-GP, and is widely distributed in various rat tissues. Studies of alkylacetyl-GP phosphohydrolase with respect to substrate specificity, pH profiles, sensitivities to temperature, and effects of detergent, ethanol, or cations indicate the activity of this enzyme can be distinguished from the activities of a nonspecific phosphomonoesterase or phosphatidate phosphohydrolase. Like alkyllyso-GP:acetyl-CoA acetyltransferase, the alkylacetyl-GP phosphohydrolase shows no notable substrate selectivities with regard to variations in alkyl chain length (C16:0 versus C18:0) at the sn-1 position or short chain acyl groups (C2:0 to C6:0, with the exception of C3:0) at the sn-2 position of the glycerol moiety. The enzymatic activity of alkylacetyl-GP phosphohydrolase is 30-90-fold higher than alkyllyso-GP:acetyl-CoA acetyltransferase in most tissues examined. Even though alkyllyso-GP is a substrate for alkyllyso-GP:acetyl-CoA acetyltransferase, it can also be degraded by alkylacetyl-GP phosphohydrolase. Thus, our findings coupled with earlier results imply that specificities of the molecular species of platelet-activating factor synthesized de novo are determined by the enzyme involved in the final step of this pathway, the dithiothreitol-insensitive alkylacetyl-G:CDP-choline cholinephosphotransferase. Furthermore, alkyl-lyso-GP:acetyl-CoA acetyltransferase appears to be the rate-limiting step in the de novo synthesis of alkylacetyl-G.     

Determination of relative values of de novo biosynthesis and salvage pathway of thymidylate formation in rat decidual tissue

A method for the determination of relative values (%) of two pathways of thymidine-5'-phosphate (dTMP) formation, e.g. via de novo biosynthesis and through thymidine reutilization (salvage pathway), is proposed. It is shown that the relative values of dTMP formation through the salvage pathway in the mesometrial part of developing decidua in pregnant rats (9-11th day of ppregnancy) are 1.5-3.4 times higher as compared to those in the antimesometrial part. When dTMP biosynthesis is suppressed by aminopterine, up to 80% of total DNA thymind is synthesized at the expense of thymidine reutilization. The incorporation of 3H-thymidine into DNA was thereby increased approximately 8-fold irrespective of the decrease in the DNA synthesis rate (approximately 2.4 times). The dependence of the relative values of the thymidine reutilization pathway on the correlation of the thymidylate synthetase and thymidine kinase activities in the tissue is discussed. The ability of the cells to reutilize thymidine is interpreted in terms of their relative resistance to the effect of folic acid antagonists.     

Subcellular localization of the pathway of de novo pyrimidine nucleotide biosynthesis in pea leaves

The subcellular distribution of the enzymes of de novo pyrimidine nucleotide biosynthesis was investigated in pea (Pisum sativum L. cv Progress No. 9) leaves. Aspartate carbamoyltransferase, the committed step of the pathway, was found to be strictly confined to the chloroplasts. Dihydro-orotase, orotate phosphoribosyl transferase, and orotidine decarboxylase activities were also found only in the plastids. The remaining enzyme of the pathway, dihydroorotate dehydrogenase, was shown to be mitochondrial.     

Concanavalin a enhances ATP resynthesis via de novo pathway in postischemic rat hearts

Acute post-ischemic cardiac failure was studied in isolated rat hearts. Xylitol, glutamine, aspartic acid and glycine were added during reperfusion resulting in no inotropic effect. Concanavalin A had a moderate inotropic effect. When concanavalin A added with xylitol, glutamine, aspartic acid and glycine, a rapid recovery of myocardial function and high-energy phosphate was achieved.     

Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270 μg/L, 61 μg/L, and 3700 μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.     

Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE

The conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-AIR (CAIR) represents an unusual divergence in purine biosynthesis: microbes and nonmetazoan eukaryotes use class I PurEs while animals use class II PurEs. Class I PurEs are therefore a potential antimicrobial target; however, no enzyme activity assay is suitable for high throughput screening (HTS). Here we report a simple chemical quench that fixes the PurE substrate/product ratio for 24 h, as assessed by the Bratton–Marshall assay (BMA) for diazotizable amines. The ZnSO₄ stopping reagent is proposed to chelate CAIR, enabling delayed analysis of this acid-labile product by BMA or other HTS methods.     

Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis

This report describes the partial characterization of the enzymatic activity responsible for the hydrolysis of acetate from 1-alkyl-2-acetyl-sn-glycerol, the immediate precursor in the de novo synthesis of PAF (platelet-activating factor or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by Ehrlich ascites cells. The highest acetylhydrolase activity for this neutral lipid was associated with the membrane fractions from Ehrlich ascites cells (> 90% of total activity); only a minimal level of activity (< 10%) was observed in the cytosol which contrasts with the cytosolic site of PAF acetylhydrolase in normal cells. Hydrolysis of 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction at pH 7.5 and 37°C gave apparent values for K_m and V_max of 45 μM and 179 nmol/min per mg protein, respectively. Hydrolysis of acetate from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction was not affected by 5 mM concentrations of Ca⁺², Mg⁺² or EDTA, but was significantly inhibited (80% reduction) by 10 mM NaF. Based on differences in both the subcellular distribution and response to inhibition by NaF, the neutral lipid acetylhydrolase does not appear to be the same enzyme that hydrolyzes acetate from platelet-activating factor. In contrast to inhibition of diacylglycerol lipase by p-chloromercuribenzoate and N-ethylmaleimide, we found no significant inhibition of acetate hydrolysis from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by either of these compounds. Also, p-nitrophenyl acetate (a nonspecific esterase substrate) failed to inhibit acetate hydrolysis of 1-[³H])hexadecyl-2-acetyl-sn-glycerol. Our studies of this enzyme would indicate that it may play an important role in regulating the levels of platelet-activating factor synthesized by the de novo pathway via hydrolysis of the immediate precursor of PAF.     

Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzi cpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.