Biomedical subjects

D L Cinti

Publications and source records attributed to D L Cinti.

At least 19 recordsLinked to original sources

PubMed1996

A program on the rise: the combined (M.D./Ph.D.) Degree Program at the University of Connecticut School of Medicine.

The Combined M.D./Ph.D. Degree Program (CDP) of the University of Connecticut School of Medicine is an intensive seven-year program that allows exceptional students simultaneously to develop both clinical and research skills. Each year the program accepts up to four highly qualified students from an applicant pool that has been increasing in both size and talent. Currently, there are 25 students in the program. Each student receives a waiver of tuition and fees for both graduate and medical school as well as a fellowship stipend of $14,700 per year for all seven years. In the initial two years the combined-degree students are enrolled in medical school and participate in laboratory research. During the next three years students focus exclusively on graduate course work and the doctoral dissertation. Following the thesis defense, they enter the final two years of medical school and are graduated with the dual degree. As of May 1995 the CDP has graduated 15 students, each of whom is currently pursuing a career in academic medicine.

Animals↗

PubMed1993

Occurrence and characterization of a dehydratase enzyme in the leek icosanoyl-CoA synthase complex.

The presence of a beta-hydroxyacyl-CoA dehydratase involved in the icosanoyl-CoA synthase (EC 2.3.1.119) complex of leek epidermis has been demonstrated using antibodies raised against the purified beta-hydroxyacyl-CoA dehydratase from rat liver. In a first step the leek icosanoyl-CoA synthase activity was measured in the presence of different amounts of this antibody, the results obtained showed a 75% inhibition of the activity using a 8:1 IgG/microsomal protein ratio, whereas only a weak diminution of the activity occurred using pre-immune IgG. The analysis of the reaction products after incubation in the presence of increasing IgG amounts showed a decrease of the fatty acids (the final product) and an accumulation of beta-hydroxy fatty acids using immune IgG, whereas no change occurred in the presence of pre-immune IgG. Moreover, the beta-hydroxyacyl-CoA dehydratase activity was strongly inhibited, whereas in the same conditions, the beta-ketoacyl-CoA reductase and the (trans-2-3) enoyl-CoA reductase activities were not affected. The protein fractions that eluted from the DEAE and Ultrogel columns containing the leek icosanoyl-CoA synthase activity were able to specifically bind the anti beta-hydroxyacyl-CoA dehydratase from rat liver. The cross-reactivity was demonstrated. In immunoblotting experiments using the same antiserum after SDS-PAGE of the purified leek icosanoyl-CoA synthase, only one of the four protein bands constituting the leek icosanoyl-CoA synthase was detected. This protein, having an apparent molecular mass of 65 kDa, could be the dehydratase component of the elongation complex.

Allium↗

PubMed1992

Evidence that beta-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin.

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.

Animals↗

PubMed1992

Depletion of rat hepatic glutathione and inhibition of microsomal trans-2-enoyl-CoA reductase activity following administration of a dec-2-ynol and dec-2-ynoic acid.

The effects of administration of dec-2-ynol and dec-2-ynoic acid on the hepatic glutathione (GSH) content and hepatic microsomal trans-2-enoyl-CoA reductase activity were examined in rat. Both compounds, when administered ip, caused a marked depletion of GSH levels and a corresponding inactivation of trans-2-enoyl-CoA reductase activity in both a time- and dose-dependent manner. The dec-2-ynoic acid caused greater hepatotoxicity than dec-2-ynol based on serum alanine transaminase activity. Based on the observations that (a) the alcohol did not interact with GSH in the presence or absence of cytosol, (b) the spectral manifestation of the interaction between GSH and the alcohol occurred only when NAD+ was added to the reaction mixture containing the cytosol and reactants, and (c) a similar absorbance spectrum was obtained following the interaction between aldehyde and GSH, it was concluded that dec-2-ynol is converted to an electrophile, dec-2-ynal, which causes depletion of GSH. The decrease in GSH content following administration of the acid appears to be due to activation of the acid to the electrophile, dec-2-ynoyl CoA, which then interacts with GSH, resulting in its depletion, based on the in vitro observations that (a) the acid did not interact with GSH in the presence or absence of cytosol, and (b) the spectral manifestation of interaction between GSH and dec-2-ynoyl CoA occurred both nonenzymatically and enzymatically in the presence of rat liver glutathione S-transferase (Sigma). Bovine serum albumin stimulated the enzymatic reaction. Comparable to the effects on GSH were the effects of dec-2-ynol, dec-2-ynal, dec-2-ynoic acid, and dec-2-ynoyl CoA on the microsomal trans-2-enoyl-CoA reductase activity in vitro. While the alcohol had no effect on the enzyme activity, its electrophilic product, the aldehyde, was a potent inhibitor. Similarly, the acid did not inhibit the enzyme activity unless the acid was present at high concentration; however, its electrophilic product, the CoA thioester, was a very potent inhibitor at very low concentration.

Acyl Coenzyme A↗

PubMed1992

The fatty acid chain elongation system of mammalian endoplasmic reticulum.

Much has been learned about FACES of the endoplasmic reticulum since its discovery in the early 1960s. FACES consists of four component reactions, requires the fatty acid to be activated in the form of a CoA derivative, utilizes reducing equivalents in the form of NADH or NADPH, is induced by a fat-free diet, resides on the cytoplasmic surface of the endoplasmic reticulum, appears to function in concert with the desaturase system and appears to exist in multiple forms (either multiple condensing enzymes connected to a single pathway or multiple pathways). FACES has been found in all tissues investigated, namely, liver, brain, kidney, lung, adrenals, retina, testis, small intestine, blood cells (lymphocytes and neutrophils) and fibroblasts, with one exception--the heart has no measurable activity. Yet, much more needs to be learned. The critical, inducible and rate-limiting condensing enzyme has resisted solubilization and purification; the purification of the other components has met with limited success. We know nothing about the site of synthesis of each component of FACES. How is each component enzyme integrated into the endoplasmic reticulum membrane? Is there a single mRNA directing synthesis of all four components or are there four separate mRNAs? How are elongation and desaturation coordinated? What is (are) the physiological regulator(s) of FACES--ADP, AMP, IP3, G-proteins, phosphorylation, CoA, Ca2+, cAMP, none of these? The molecular biology of FACES is only in the fetal stage of development. We are only scratching the surface--it is an undiscovered country.

Animals↗

PubMed1991

Do rat kidney cortex microsomes possess the enzymatic machinery to desaturate and chain elongate fatty acyl-CoA derivatives?

Rat kidney cortex microsomal preparations were unable to catalyze delta 9, delta 6 and delta 5 desaturation of stearoyl-coenzyme A (CoA), linoleoyl-CoA and dihomo-gamma-linolenoyl-CoA, respectively. The kidney cortex microsomal fraction, however, did catalyze the malonyl-CoA dependent fatty acyl-CoA elongation. The biochemical properties of palmitoyl-CoA elongation were studied as a function of protein concentration, time, reduced nicotinamide adenine dinucleotide phosphate (NADPH), malonyl-CoA and substrate concentrations; of the substrates investigated, delta 6,9,12-18:3 was the most active. Unlike what was observed in the hepatic system, a high-carbohydrate, fat-free diet did not induce kidney fatty acid chain elongation. All intermediate kidney cortex microsomal reactions, i.e., beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase activities, were significantly higher (greater than one order of magnitude) than the condensing enzyme activity, suggesting that the rate-limiting step in total elongation is the initial condensation reaction. Contrary to other reports, the results suggest that the kidney cannot synthesize arachidonic acid needed for eicosanoid production.

Acyl Coenzyme A↗

PubMed1991

Decreased long-chain fatty acyl CoA elongation activity in quaking and jimpy mouse brain: deficiency in one enzyme or multiple enzyme activities?

Using long-chain fatty acyl CoAs (arachidoyl CoA and behenoyl CoA), a decrease in overall fatty acid chain elongation activity was observed in the quaking and jimpy mouse brain microsomes relative to controls. Arachidoyl CoA (20:0) and behenoyl CoA (22:0) elongation activities were depressed to about 50% and 80% of control values in quaking and jimpy mice, respectively. Measurement of the individual enzymatic activities of the elongation system revealed a single deficiency in enzyme activity; only the condensation activity was reduced to the same extent as total elongation in both quaking and jimpy mice. The activities of the other three enzymes, beta-ketoacyl CoA reductase, beta-hydroxyacyl CoA dehydrase, and trans-2-enoyl CoA reductase, in both mutants were similar to the activities present in the control mouse. In addition, the activities of these three enzymes were more than two to three orders of magnitude greater than the condensing enzyme activity in all three groups, establishing that the condensing enzyme catalyzes the rate-limiting reaction step of total elongation. When the elongation of palmitoyl CoA was measured, only a 25% decrease in total elongation occurred in both mutants; a similar percent decrease in the condensation of palmitoyl CoA also was observed. The activities of the other three enzymes were unaffected. These results support the concept of either multiple elongation pathways or multiple condensing enzymes.

Acetyltransferases↗

PubMed1990

Enzyme site-specific changes in hepatic microsomal fatty acid chain elongation in streptozotocin-induced diabetic rats.

The hepatic microsomal fatty acid chain elongation of palmitoyl-CoA and gamma-linolenoyl-CoA was diminished by 40-50% in male Sprague-Dawley rats made diabetic for 2 and 4 weeks following the intravenous administration of a single dose (65 mg/kg) of streptozotocin. Analysis of the activities of the four enzymatic components showed that only one enzyme, the condensing enzyme, which catalyzes the initial and rate-limiting step in chain elongation, was altered by the diabetic state. Both chain elongation and condensation activities were depressed to the same extent, whereas beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase activities were the same as the values obtained with non-diabetic controls. 2 week administration of 10 units of insulin per day to rats which were diabetic for a 2-week period resulted in the reversal of the reduced palmitoyl-CoA elongation and condensation activities to control values. However, neither the condensation nor the elongation of gamma-linolenoyl was reversed by the insulin treatment. These results support the notion of multiple condensing enzymes or chain elongation systems.

Acyl Coenzyme A↗

PubMed1989

Evidence for two separate beta-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat.

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the beta-ketoacyl derivative. Employing beta-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported beta-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent beta-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two beta-ketoacyl CoA reductases, one using NADH and the other NADPH.

Acyl Coenzyme A↗

PubMed1989

Evidence for two separate beta-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Topography of rat hepatic microsomal enzymatic components of the fatty acid chain elongation system.

The orientation of the condensing enzyme, the beta-hydroxyacyl-CoA dehydrase, and the trans-2-enoyl CoA reductase within the rat liver microsomal membrane was investigated by the use of impermeant inhibitors of enzyme activity: trypsin, chymotrypsin, subtilisin, mercury-dextran, and anti-beta-hydroxyacyl-CoA dehydrase IgG. The activity of the condensing enzyme was inhibited more than 70% by various proteases and was completely inhibited by 80 microM mercury-dextran. Similar results were obtained for the trans-2-enoyl-CoA reductase activity. On the other hand, in the absence of detergent, proteases inhibited beta-hydroxyacyl-CoA dehydrase activity by 25-40%, while in the presence of detergent the inhibition increased to 65-90%. Furthermore, anti-beta-hydroxyacyl-CoA dehydrase IgG, which in the absence of detergent produced no inhibition, in the presence of detergent inhibited beta-hydroxyacyl-CoA dehydrase activity by more than 80%; under identical conditions, preimmune IgG caused a 13% inhibition. Microsomes used throughout this study displayed greater than 90% latency with respect to mannose-6-phosphatase activity, indicating that the microsomes were intact. Latency was not affected by the proteases, by mercury-dextran, or by the presence of the enzyme assay components. These results suggest that both the condensing enzyme and the reductase are present on the cytoplasmic surface of the membrane, whereas the beta-hydroxyacyl-CoA dehydrase is embedded in the microsomal membrane.

Acyl-CoA Dehydrogenases↗

PubMed1989

Disruption of rat hepatic microsomal electron transport chains by the selenium-containing anti-inflammatory agent Ebselen.

The influence of Ebselen, an organoselenium anti-inflammatory agent, on the two electron transport chains present in rat liver microsomes has been studied. At low micromolar concentrations, Ebselen markedly inhibited the flow of reducing equivalents from NADPH-cytochrome P450 reductase to both its natural electron acceptor, cytochrome P450, and its artificial electron acceptor, cytochrome c. Similarly, the microsomal NADH-cytochrome c reductase system consisting of cytochrome b5 and its flavoprotein, NADH-cytochrome b5 reductase, was also significantly inhibited by Ebselen. The inhibition appears to be due to the inability of the reduced pyridine nucleotide to transfer electrons to the flavin (FAD and/or FMN) in the flavoprotein reductase. This was shown with the purified NADPH-cytochrome P450 reductase, which in the presence of Ebselen was not converted to the semiquinone form following the addition of NADPH. The addition of Ebselen to a suspension of hepatic microsomes from either untreated or phenobarbital-treated rats did not result in any spectral change characteristic of type I, type II, or reverse type I.

Animals↗

PubMed1989

Action of Ebselen on rat hepatic microsomal enzyme-catalyzed fatty acid chain elongation, desaturation, and drug biotransformation.

In the previous study, the organoselenium-containing anti-inflammatory agent, Ebselen, was found to disrupt both hepatic microsomal NADH- and NADPH-dependent electron transport chains. In the current investigation, we focus on the action of Ebselen on three separate metabolic reactions, namely, fatty acid chain elongation, desaturation, and drug biotransformation, which utilize reducing equivalents via these microsomal electron transport pathways. Both NADH-dependent and NADPH-dependent chain elongation reactions showed (i) that the condensation step was inhibited by Ebselen; all three substrates, palmitoyl CoA (16:0), palmitoleoyl CoA (16:1), and gamma-linolenyl CoA (18:3), were differentially affected by Ebselen; for example, the apparent Ki's of Ebselen for the condensation of 16:0, 16:1, and 18:3 in the absence of bovine serum albumin (BSA) preincubation were 7, 14, and 34 microM, and those in the presence of BSA preincubation were 35, 62, and 150 microM, respectively, supporting earlier data for multiple condensing enzymes; (ii) that the beta-ketoacyl CoA reductase-catalyzed reaction step which appears to receive electrons, at least in part, from the cytochrome b5 system, was also markedly inhibited by varying Ebselen concentrations; and (iii) that similar results were obtained with the dehydrase and the enoyl CoA reductase. Hence, each of the four component steps was significantly inhibited by Ebselen. Another important fatty acid biotransformation reaction, delta 9 desaturation of stearoyl CoA to oleoyl CoA, was significantly inhibited (90%) by 30 microM Ebselen. This effect appeared to be directly related to the NADH-dependent electron transport chain rather than to a direct action on the desaturase enzyme. Last, Ebselen also inhibited both aminopyrine and benzphetamine N-demethylations, two cytochrome P450-catalyzed reactions, in untreated rats, in rats on a high carbohydrate diet, and in phenobarbital-treated rats.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

PubMed1989

Spectrophotometric assay for the condensing enzyme activity of the microsomal fatty acid chain elongation system.

A rapid and simple spectrophotometric method was developed to measure the activity of the condensing enzyme component of the microsomal fatty acid chain elongation system. The intermediate product of the condensation reaction is the beta-ketoacyl CoA which exists in two tautomeric forms, i.e., keto and enol. The addition of bovine serum albumin (BSA) to a cuvette cell containing a beta-ketoacyl CoA derivative resulted in the formation of a 303-nm absorbance peak, characteristic of enolate formation. The beta-ketoacyl CoAs with carbon chain length of 6 to 18 interacted with BSA to produce the 303-nm peak; acetoacetyl CoA was the only beta-keto compound tested which did not interact with BSA to produce the peak. Other compounds which were unaffected by BSA included CoA, free beta-keto acid, beta-hydroxyacyl CoA, acyl CoA, trans-2-enoyl CoA, and malonyl CoA. BSA could not be replaced by ovalbumin; furthermore, denatured (boiling) BSA could not induce the 303-nm peak. The specific activity of the condensing enzyme measured by the spectrophotometric method compares favorably with the activity obtained by the radioactive method. The apparent extinction coefficient (epsilon) for the absorbance peak generated by the beta-keto thioester varied from 5 to 30 mM-1 cm-1 depending on the beta-keto derivative. The spectrophotometric procedure can be used in the determination of the condensing enzyme activity in not only hepatic microsomes but also in kidney and brain microsomes both of which have significantly lower activity. The advantages of the novel method over the radioactive method are that (i) it does not involve the use of radioactive compounds, (ii) it is much less cumbersome and significantly less costly, and (iii) it is rapid and easy to perform.

Animals↗

PubMed1988

Dual action of 2-decynoyl coenzyme A: inhibitor of hepatic mitochondrial trans-2-enoyl coenzyme A reductase and peroxisomal bifunctional protein and substrate for the mitochondrial beta-oxidation system.

The present study was designed to determine the action of the 2-acetylenic acid thioester on mitochondrial fatty acid chain elongation and beta-oxidation. Addition of 2-decynoyl CoA to a rat liver mitochondrial suspension resulted in a significant stimulation of the rate of oxidation of NADPH and NADH. This enhanced oxidation rate was not due to the mitochondrial trans-2-enoyl CoA reductase-catalyzed conversion of the 2-acetylenic acid thioester to the saturated product, decanoate, as measured by gas-liquid chromatography. On the contrary, the mitochondrial trans-2-enoyl CoA reductase activity was markedly inhibited by the 2-acetylenic acid derivative, as evidenced by the decrease in the reduction of trans-2-decenoyl CoA to decanoic acid. Incubation of the mitochondrial fraction with either NADPH or NADH and 2-decynol CoA resulted in the gas chromatographic identification of three products: beta-ketodecanoate, beta-hydroxydecanoate, and trans-2-decenoate. In the absence of reduced pyridine nucleotide, a single product was formed and identified as beta-ketodecanoate. Confirmation of the identity of this product was obtained by the observation of the formation of the Mg2+-enolate complex (303-nm absorbance peak). These results suggest that, although the 2-decynoyl CoA is an inhibitor of mitochondrial trans-2-enoyl CoA reductase activity, it is a substrate for the mitochondrial trans-2-enoyl CoA hydratase (crotonase). This was confirmed by incubation of 2-decynoyl CoA with commercially purified liver mitochondrial crotonase. The beta-ketodecanoate is formed in a two-step process: hydration of the 2-decynoyl CoA to an unstable enol intermediate which undergoes rearrangement to the beta-ketodecanoyl CoA. Interestingly, although the mitochondrial crotonase can utilize the 2-acetylenic acid thioesters, this was not the case for the peroxisomal bifunctional hydratase which was markedly inhibited by varying concentrations of 2-decynoyl CoA.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1987

Biochemical and immunological identity of the hepatic peroxisomal and microsomal trans-2-enoyl CoA hydratase bifunctional protein.

In the present study, the hepatic microsomal and peroxisomal bifunctional trans-2-enoyl CoA hydratases were isolated and purified from rats treated with 2% di-(2-ethylhexyl)phthalate for 8 days. These two enzymes (microsomal and peroxisomal) were purified with the identical purification procedures and had identical molecular masses of 76 kDa. A single band was observed on an electrophoretic gel of an equimixture of the two proteins. Both preparations had identical pI's of 8.6 and pH optima of 6.0 for the dehydrogenase (reductase) and 7.5 for the hydratase activity. Two-dimensional gel analysis of an equimixture of the two preparations showed only one band. Ouchterlony double-diffusion analysis showed that an antibody raised against the purified microsomal enzyme interacted at a point with the peroxisomal enzyme, indicating immunologic identity. Western blot analysis demonstrated that the antibody formed a single band with total microsomal and peroxisomal fractions. The antibody inhibited the enzymatic activities of both preparations in a similar manner. Interestingly, the antibody had a markedly greater inhibitory effect on the reductase activity of the two enzyme preparations, and a much less inhibitory effect on the hydratase activity, suggesting that the antigenic determinants reside at or near the catalytic site of the reductase portion of the protein. These results suggest that the microsomal and peroxisomal bifunctional proteins are identical.

Animals↗

PubMed1987

Source of the hepatic microsomal trans-2-enoyl CoA hydratase bifunctional protein: endoplasmic reticulum or peroxisomes.

The present study was designed to investigate the hepatic localization of the microsomal bifunctional trans-2-enoyl CoA hydratase. Despite the low activity (less than 10%) of peroxisomal marker enzymes in isolated hepatic microsomes (acyl CoA oxidase (this study), catalase, and urate oxidase (L. Cook, M. N. Nagi, J. Piscatelli, T. Joseph, M. R. Prasad, D. Ghesquier, and D. L. Cinti, 1986, Arch. Biochem. Biophys. 245, 24-26), additional evidence in this study suggests that the microsomal enzyme is derived from peroxisomes. For example, the microsomal hydratase activity was associated with the ribosomal fractions but not with the smooth endoplasmic reticulum. In addition, when an extract of the peroxisomal enzyme was incubated with either free ribosomes or membrane-bound ribosomes, marked binding was observed with each of the fractions. Furthermore, the ease of release of the bifunctional enzyme from both free ribosomes and membrane-bound ribosomes by only KCl suggests that the bound enzyme is not a nascent protein. Labeling of liver tissue from DEHP-treated rats with rabbit immune IgG made to the purified microsomal hydratase followed by gold conjugated goat anti-rabbit IgG suggested a single subcellular site for the bifunctional hydratase--the peroxisomal organelle.

Adsorption↗

PubMed1986

Do rat hepatic microsomes contain multiple NADPH-supported fatty acid chain elongation pathways or a single pathway?

[2-14C]-trans-2-hexadecenoyl CoA (16:1) and [2-14C]-trans-2-cis-8,11,14-eicosatetraenoyl CoA (20:4) were chemically synthesized and employed as competitive substrates for the liver microsomal trans-2-enoyl CoA reductase component of the fatty acid chain elongation system. Both 7.5 microM and 15 microM 20:4 competitively inhibited the reduction of 16:1 CoA to palmitoyl CoA. In addition, the reduction of both substrates was identically inhibited to the same extent by the acetylenic derivative, dec-2-ynoyl CoA. Furthermore, trypsin, chymotrypsin and subtilisin inhibited trans-2-enoyl CoA reductase activity when three different substrates were employed--16:1, 20:4 and trans-2-cis-11-octadecadienoyl CoA (18:2). These results are consistent with the hypothesis of multiple condensing enzymes connected to a single elongation pathway.

Acyl-CoA Dehydrogenases↗