Presence Of N-ethylmaleimide Research Articles

Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes.

Whereas there is biological evidence that the reductive cleavage of disulfide bonds is critical for the activation of endocytosed macromolecules such as toxins, immunotoxins, and other drug carriers, virtually nothing is known about the specifics of this cleavage. To study this process, a model compound was synthesized in which a radioiodinated tyramine was linked through a disulfide bond to an undegradable carrier, poly(D-lysine), known to be efficiently endocytosed. Cultured Chinese hamster ovary cells were pulse-labeled with this probe, and the disulfide cleavage was measured as released acid-soluble radioactivity at different times of chase. Pulse-labeled cells were also subjected to subcellular fractionation to identify intracellular structures associated with disulfide cleavage. Cleavage began without lag, amounted to about approximately 7% of the initial cell-bound radioactivity in the first hour and continued for more than 6 h. It was abolished in the presence of N-ethylmaleimide. When sulfhydryl groups present at the cell surface were blocked with cell-impermeant sulfhydryl reagent, the initial phase of disulfide cleavage was inhibited, indicating that cleavage began at the cell surface. A long-lasting intracellular phase of disulfide cleavage began after about approximately 30 min of chase. Subcellular fractionation and kinetic analysis indicated that neither lysosomes nor endosomes were participating in that phase, leaving the Golgi apparatus as the most probable site of endocytic disulfide cleavage.

Association of simian virus 40 small-t antigen with the 61-kilodalton component of a cellular protein complex

Two cellular proteins, 61 and 37 kDa, are found in association with the simian virus 40 (SV40) small-t antigen. Fractionation in standard chromatography systems showed that these proteins were associated with one another in uninfected cells, suggesting that the small-t antigen may bind the complex as a whole and not each individual protein independently. In the presence of N-ethylmaleimide, the 37-kDA protein was selectively released from immune complexes, leaving the small-t antigen and 61-kDa protein in association. This result suggests that the small-t antigen may bind only the 61-kDa protein and that the 37-kDa protein may be associated with immune complexes by virtue of its association with the 61-kDa cellular protein.

Evidence for active H+ secretion by rat alveolar epithelial cells.

A plasma membrane proton-translocating adenosinetriphosphatase (ATPase) has been identified in rat alveolar pneumocytes in primary culture using the pH-sensitive fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Intracellular pH (pHi) was acutely lowered by NH3 prepulse in HCO3(-)-free medium buffered with 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and its recovery was measured thereafter under control conditions, in the presence of amiloride to inhibit Na(+)-H+ antiport, and in the presence of N-ethylmaleimide (NEM), a plasma membrane H(+)-ATPase inhibitor. Initial rate of pHi recovery was reduced by 67% in the presence of amiloride, 52% in the presence of NEM, and 96% in the presence of both. Recovery was decreased but not abolished in Na(+)-free buffer, was essentially abolished when NEM was present in the absence of Na+, and was also abolished by addition of the metabolic inhibitor KCN in glucose- and Na(+)-free medium. These data suggest that alveolar epithelial cells possess a plasma membrane H(+)-ATPase. In Na(+)-containing buffer at pH 7.4, steady-state pHi was 7.50. This value was unaffected by amiloride but decreased to 7.01 in the presence of NEM, suggesting active H(+)-ATPase and inactive Na(+)-H+ antiport at steady-state pHi. We conclude that this plasma membrane proton-translocating ATPase in alveolar pneumocytes may be an important mechanism contributing to regulation of steady-state pHi, recovery from acute intracellular acidification, and modulation of extracellular alveolar fluid pH.

Sulfation in male reproductive organs bull and boar testis phenol sulfotransferases

Phenol sulfotransferases (PST) from bull and boar testis were partially purified and characterized. A single form of PST adsorbed on DEAE-cellulose was found in the bull testis, whereas from boar testis two different peaks of PST activity were separated. The bull testis PST and both boar testis enzymes were active with p-nitrophenol and adrenalin. They all showed higher affinity to pNP than to adrenalin and were inhibited by these substrates at higher concentrations. Their optimal pH was at 8.5. Bull testis PST and boar PST II which were adsorbed on DEAE-cellulose were thermostable, whereas boar PST I was thermolabile. Those three PST forms differed in sensitivity to 2,6-dichloro-4-nitrophenol (DCNP), N-ethyl maleimide (NEM), lodoacetamide (IAA) and phenylglyoxal (PG). Bull and boar PST II were more rapidly inactivated in the presence of DCNP than boar PST I. In the presence of NEM, the —SH groups reagent, the bull phenol sulfotransferase and boar PST I lost their activity, whereas the activity of boar PST increased. Also iodoacetamide, another —SH group modificator, raised boar PST II activity and decreased boar PST I activity. DTT, which protects thiol groups, had an opposite effect on the enzymes studied than NEM. Phenylglyoxal, a reagent specific for arginine residues inhibited bull testis PST and both boar phenol sulfotransferases. Substrate protection experiments were also performed to determine the localization of reactive groups in bull and boar testis phenol sulfotransferases.

Effects of N-ethylmaleimide on 5-hydroxytryptamine transport and sodium content in rabbit platelets.

1. The present study analysed the mechanism underlying the inhibitory action of N-ethylmaleimide (NEM) on the 5-hydroxytryptamine (5-HT) uptake by blood platelets. 2. Rabbit platelets suspended in protein-free buffer were first preincubated for 45 min in the absence and presence of NEM (20 to 160 microM) or ouabain (0.5-2.0 microM) and then either analysed for their Na+ and K+ content or incubated (15s) with various concentrations of [3H]-5-HT (0.13-4.03 microM) to determine Km and Vmax for 5-HT uptake. 3. Both NEM and ouabain produced concentration-dependent decreases in Vmax with IC50 values of 52 and 0.58 microM, respectively. Neither drug changed Km significantly. 4. Both NEM and ouabain increased the Na+ and decreased the K+ content of platelets in a concentration-dependent manner. 5. There was a linear correlation between Vmax (expressed in % of control) and the reciprocal cellular Na+ content, with the results for both drugs falling onto one and the same regression line (r = 0.992; n = 8). This regression showed that an increase in Na+ content by 69% sufficed to reduce Vmax by 50%. 6. At concentrations that reduced 5-HT uptake by about 60%, neither NEM nor ouabain altered the potency of imipramine for inhibition of 5-HT uptake. 7. Hence, NEM inhibits 5-HT transport by inhibiting the Na+/K+-ATPase and not by a direct interaction with the 5-HT carrier. The consequential increase in the intracellular Na+ concentration reduces the transmembrane Na+ gradient and, therefore, hinders 5-HT inward transport. This action of the drug does not affect the ability of the carrier to bind 5-HT or imipramine.

Mammalian Progesterone Receptor Shows Differential Sensitivity to Sulfhydryl Group Modifying Agents When Bound to Agonist and Antagonist Ligands

Modulation of calf uterine progesterone receptor (PR), in relation to its binding to synthetic steroids with known agonist (R5020) and antagonist (RU486) properties, was studied in the presence of iodoacetamide (IA), N-ethylmaleimide (NEM), beta-mercaptoethanol (MER), and dithiothreitol (DTT). Pretreatment of uterine cytosol at 4 degrees C with NEM (4-10 mM) reduced the binding of [3H]RU486 to PR by 40%, but [3H] R5020 binding was completely abolished. Whereas IA (2-10 mM) treatment did not affect [3H]RU486 binding, [3H]R5020 binding was totally eliminated. DTT or MER increased the binding of both steroids slightly (15%). [3H]R5020- or [3H]RU486-receptor complexes (Rc) migrated in the 8 S region and were eliminated upon pretreatment with NEM. At 23 degrees C, DTT increased the amount of 4 S [3H]R5020-Rc, but had no effect on the [3H]RU486-Rc. In the control, [3H]RU486 binding to the 8 S PR could be competed with radioinert R5020 or RU486, but R5020 failed to compete in the presence of IA. The heat-treated [3H]R5020- and [3H]RU486-Rc showed reduced binding to DNA-cellulose in the presence of NEM and IA. The results of our study suggest that SH group modifications differentially influence the properties of mammalian PR complexed with either R5020 or RU486. In the presence of IA, the [3H]RU486-Rc remained in the 8 S form when incubated at 23 degrees C, indicating that RU486 binding causes conformational changes in PR which are distinct from those that result upon R5020 binding.

Induction of human basophil histamine release by a novel protein kinase C activator, sn‐1,2‐isopropylidene‐3‐decanoyl‐glycerol (IpOCOC9): Partial characterization of secretagogue characteristics

Human leukocytes were found to release histamine at exposure for the synthetic glyceride derivative sn-1,2-isopropylidene-3-decanoyl-glycerol (IpOCOC9). The following characteristics for the IpOCOC9-induced basophil histamine release were recorded. A. In the order of 25% of the cellular histamine content was extruded at 206 mumol/l and 45% at 690 mumol/l of the compound, respectively. B. Removal of extracellular Ca2+ variably affected IpOCOC9-triggered release. C. The presence of N-ethylmaleimide (10 mumol/l) or p-bromophenacylbromide (10 mumol/l) markedly reduced IpOCOC9-induced histamine release. D. The time course of the release triggered by IpOCOC9 was intermediate to those characterizing the release triggered by 4 beta-phorbol 12-myristate 13-acetate (PMA) and by formyl-methionyl-leucyl-phenylalanine (FMLP). E. Cells desensitized to IgE-receptor-mediated stimulation were hyperresponsive to stimulation with IpOCOC9. F. Cells treated with a low concentration of 2-deoxyglucose were not hyperresponsive to IpOCOC9. These data show that IpOCOC9, a PMN/leukocyte protein kinase C stimulator, acts as a non-cytotoxic secretagogue for human basophils with a mode of action which in some, but not all respects, mimics that of PMA. In particular, IpOCOC9-triggered release resembles that reported by other authors for hyperosmolar triggering of release by mannitol.

Role of nonprotein thiols in enzymatic reduction of 2-nitroimidazoles

The role of nonprotein thiols (NPSH) in the enzymatic reduction of the nitro function in 2-nitroimidazoles (2-NI) has been investigated. The addition of NPSH has been shown previously to protect cells from the hypoxic cytotoxicity of 2-NI, whereas depletion of NPSH enhances the hypoxic cytotoxicity. In this report, we have investigated the effects of thiol depleting agents, N-ethylmaleimide (NEM) and diethyl maleate (DEM), on the enzymatic reduction of the nitro group. Cytosolic and microsomal fractions of rat hepatic tissue and xanthine oxidase were employed as sources of nitro reductases. Addition of NPSH caused an enhancement in the reduction of the nitro group of 2-NI; cysteine was significantly more effective than glutathione (GSH) in stimulating the enzymatic reduction. The reduction of the nitro function was decreased markedly in the presence of NEM or DEM. Addition of cysteine or GSH reversed the inhibition with NEM. Both NEM and DEM also attenuated the enhancement of reduction observed after the addition of NPSH. These results suggest that the addition of NPSH facilitates the reduction of the nitro function to the reduced intermediates that may be inactivated by an excess of NPSH, whereas the depletion of NPSH allows the accumulation of the toxic nitro radicals causing increased cytotoxicity.

Lamin A is not synthesized as a larger precursor polypeptide

Isolation of rat liver nuclei in the presence of N-ethylmaleimide (NEM) led to the recovery in the final nuclear matrix of a higher molecular weight form of lamin A. The 2 kDa larger form was identified as lamin A by isoelectric point determination, recognition by an anti-lamin A and C monoclonal antibody and peptide mapping using V8 protease and N-chlorosuccinimide. The 2 kDa extension was tentatively localized to the carboxy-terminus of lamin A. Pulsechase labeling and immunoprecipitation studies using baby hamster kidney cells showed that lysis of the cells in the presence of NEM allowed the recovery of a stable higher molecular weight form of lamin A. We conclude from these results that NEM prevented the degradation of the native form of lamin A previously thought to represent a higher molecular weight transient precursor form.

Primary structure around the lipoate-attachment site on the E2 component of bovine heart pyruvate dehydrogenase complex.

Bovine heart pyruvate dehydrogenase complex was acetylated by using [3-14C]pyruvate in the presence of N-ethylmaleimide, with approx. 1 mol of acetyl groups being incorporated per mol of E2 polypeptide. After peptic digestion, lipoate-containing peptides were purified by high-voltage electrophoresis and ion-exchange and reverse-phase h.p.l.c. The amino acid sequence around the lipoic acid-attachment site of E2 was determined by automated Edman degradation. Acetylation of a lipoate cofactor bound to a lysine residue was verified by fast-atom-bombardment m.s.

Tac antigen forms disulfide-linked homodimers.

Interleukin 2 (IL-2) is responsible for stimulating T-cell proliferation via interaction with specific, high-affinity membrane receptors. Although an IL-2-binding protein has been identified by virtue of its reactivity with a monoclonal antibody that competes with IL-2 for binding (anti-Tac), the complete and precise structure of functional IL-2 receptors is still unknown. To define further the composition of IL-2 receptors, both IL-2 itself and anti-Tac were used as ligands to adsorb membrane proteins for analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). A variety of experimental approaches yield results indicating that the Tac antigen, which migrates as a single protein on NaDod-SO4/PAGE under reducing conditions, is also expressed as disulfide-linked homodimers and oligomers. Examined under nonreducing conditions, both activated normal human T cells and cell lines from patients with adult T cell leukemia (HUT-102; MT-1) express Tac antigen homodimers (Mr 105,000) in addition to monomers (Mr 54,000). Formation of the disulfide bond is not a consequence of the experimental procedures used to isolate the proteins for analysis, inasmuch as identical results are obtained when the receptor proteins are iodinated and extracted in the presence of N-ethylmaleimide or prepared for electrophoresis in the absence of heat denaturation. Accordingly, these findings point to Tac antigen associating with itself preferentially. The physiologic significance of homodimer and oligomer formation, especially as it relates to the formation of high-affinity IL-2 receptors, is presently unknown.

Enhancement of the Rectal Absorption of Sodium Ampicillin by N-Acylamino Acids in Rats

The promoting efficacies of N-acyl derivatives of amino acids on the rectal absorption of sodium ampicillin were investigated using the rat rectal loop technique. N-Acyl derivatives with longer carbon chains in the acyl moiety showed a greater promoting potency. The promoting potencies of N-acyl derivatives of phenylglycine and phenylalanine were greater than those of glycine and alanine derivatives when compared at the same length of carbon chain in their acyl moieties. The promoting action of N-acylamino acids was not influenced by the presence of N-ethylmaleimide or ouabain. The promoting potencies of N-acylamino acids were depressed in the presence of calcium chloride in the rectal loop. The contribution of the calcium ion sequestration capacity of N-acylamino acids to their promoting efficacies is discussed.

Two distinct DNA ligases from Drosophila melanogaster embryos

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'-phosphoryl and 3'-hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg 2+ for activity. The K m for ATP is 2.7 × 10 −6 M for DNA ligase I, and 3.0 × 10 −5 M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N-ethylmaleimide. DNA ligase I is active at a low salt concentration (0–30 mM KCl), but DNA ligase II is active at high salt concentrations (50–100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase-AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637–10645] seem to be the same enzyme.

Molecular distinction between calf heart and brain muscarine receptors: Different N-ethylmaleimide modulation of agonist binding

Muscarinic acetylcholine receptors in calf heart and forebrain membranes were identified by binding of 1-[benzilic-4,4′- 3H] quinuclinyl benzilate ([ 3H]QNB). We were able to solubilise these receptors with a yield of 50% of the proteins by treatment of the membranes with digitonin. The existence of two or more receptor subclasses with different agonist affinity in the heart membranes was evidenced by the shallow carbachol/[ 3H]QNB competition binding curves. The receptors displayed only low agonist affinity, in the presence of GTP as well as after solubilisation. The alkylating reagent N-ethylmaleimide (NEM) caused a 70-fold increase in agonist affinity for the solubilised receptors whereas GTP was ineffective. A similar difference in affinity was observed for the membranes when agonist competition curves in the presence of NEM were compared to those in the presence of GTP. NEM caused only a 2- to 3-fold increase of the agonist affinity for solubilised brain cortex membranes. These data suggest that heart and brain muscarine receptors are structurally different.

Euglena gracilis chloroplast elongation factor Tu. Interaction with guanine nucleotides and aminoacyl-tRNA.

The interaction of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis with guanine nucleotides and aminoacyl-tRNA has been investigated. The apparent dissociation constant at 37 degrees C for the EF-Tuchl X GDP complex is about 3 X 10(-7) M and for the EF-Tuchl X GTP complex, it is about 1 order of magnitude higher. The sulfhydryl modifying reagent N-ethylmaleimide severely inhibits the polymerization activity of Euglena EF-Tuchl. In the presence of N-ethylmaleimide, the dissociation constant for the modified EF-Tuchl X GDP complex is increased by an order of magnitude. Conversely, both GDP and GTP protect EF-Tuchl from the modification. The polymerization activity of EF-Tuchl is also sensitive to the antibiotic kirromycin. In the presence of kirromycin, the apparent dissociation constant for the EF-Tuchl X GTP complex is lowered 10-fold. The interaction of aminoacyl-tRNA with EF-Tuchl was investigated by examining the ability of EF-Tuchl to prevent the spontaneous hydrolysis of Phe-tRNA and by gel filtration chromatography. The binding of aminoacyl-tRNA to EF-Tuchl occurs only in the presence of GTP indicating the formation of the ternary complex EF-Tuchl X GTP X Phe-tRNA. The effect of kirromycin on the interaction was also investigated. In the presence of kirromycin, no interaction between EF-Tuchl and Phe-tRNA is observed, even in the presence of GTP.

Cysteamine depletes cystinotic leucocyte granular fractions of cystine by the mechanism of disulphide interchange.

Cystinotic lysosome-rich leucocyte granular fractions, loaded with [35S]cystine, were exposed to different cystine-depleting agents. During a 30 min incubation at 37 degrees C, untreated cystinotic granular fractions lost negligible [35S]cystine when corrected for lysosome rupture. Granular fractions exposed to 0.1 mM-cysteamine lost 64% of their initial cystine, and hexosaminidase activity was decreased by 10%. This was accompanied by the formation of high concentrations of [35S]cysteine-cysteamine mixed disulphide within the granular-fraction pellet, and, in the presence of N-ethylmaleimide, increasing amounts of [35S]cysteine-N-ethylmaleimide adduct outside the granular fraction. In separate experiments, [35S]cystine exited cystinotic leucocyte lysosomes at a negligible rate (half-times 199 and 293 min), but [35S]cysteine-cysteamine mixed disulphide exhibited substantial egress (half-times 66 and 88 min) and was recovered intact outside the granular-fraction pellet. We conclude that cysteamine depletes lysosomes of cystine by participating in a thiol-disulphide interchange reaction to produce cysteine and cysteine-cysteamine mixed disulphide, both of which traverse the cystinotic leucocyte lysosomal membrane.

Adenine nucleotide and phosphate transport systems of mitochondria. Relative location of sulfhydryl groups based on the use of the novel fluorescent probe eosin-5-maleimide.

Eosin-5-maleimide is impermeable to the inner mitochondrial membrane, exhibiting essentially no reactivity with matrix glutathione or with beta-hydroxybutyrate dehydrogenase located on the matrix surface of the inner membrane. In intact mitochondria, eosin-5-maleimide is unreactive with the ADP/ATP antiporter even under conditions which promote maximal labeling by N-[3H]ethylmaleimide (i.e., ADP present). However, eosin-5-maleimide readily labels the ADP/ATP antiporter in "inverted" inner membrane vesicles even in the presence of N-ethylmaleimide. Labeling is prevented if the vesicles are prepared from mitochondria pretreated with carboxyatractyloside. In contrast to the ADP/ATP antiporter, essential sulfhydryl groups of the Pi/H+ symporter are accessible to eosin-5-maleimide in intact mitochondria with optimal inhibition of phosphate transport being observed at 25 degrees C. Eosin-5-maleimide also prevents labeling of the Pi/H+ symporter by N-[3H]ethylmaleimide. These results show that essential sulfhydryl groups of the ADP/ATP antiporter and the Pi/H+ symporter have differing reactivities and locations in functionally intact mitochondria. With respect to eosin-5-maleimide, sulfhydryl groups of the ADP/ATP carrier occur in two distinct classes, both of which are inaccessible in intact mitochondria. Only one class, depending on conditions, can be exposed in submitochondrial particles. In contrast, sulfhydryl group(s) of the Pi/H+ symporter behave as a single reactive class which is readily accessible in mitochondria at 25 degrees C.

Stoichiometry of Proton Translocation Coupled to Substrate Oxidation in Plant Mitochondria

The proton translocation coupled to the electron flux from succinate, exogenous NADH, and NAD(+)-linked substrates (malate and isocitrate) to cytochrome c and to oxygen was studied in purified potato (Solanum tuberosum) mitochondria using oxygen and ferricyanide pulse techniques. In the presence of valinomycin plus K(+) (used as a charge compensating cation), optimum values of H(+)/2 e(-) were obtained when low amounts of electron acceptors (oxygen or ferricyanide) were added to the mitochondria (1-2 nanogram [2 e(-)] equivalents per milligram protein). The stoichiometry of proton translocation to electron flux was unaffected in the presence of N-ethylmaleimide, an inhibitor of the Pi/H(+) symport. With succinate as substrate, H(+)/2 e(-) ratios were 4.0 +/- 0.2 and 3.7 +/- 0.3 with oxygen and ferricyanide as electron acceptors, respectively. With exogenous NADH, H(+)/2e(-) ratios were 4.1 +/- 0.9 and 3.4 +/- 0.2, respectively. The proton translocation coupled to the oxidation of NAD(+)-linked substrates (malate, isocitrate) was dependent upon the presence of adenylates (ADP, AMP, or ATP). For malate (+ glutamate) oxidation the observed H(+)/2 e(-) ratios were increased from 3.6 +/- 2.2 to 6.5 +/- 0.5 in the presence of 20 micromolar ADP.

Fatty acid elongation activity in fibroblasts from patients with adrenoleukodystrophy (ALD).

The activities of microsomal fatty acid elongation and cytoplasmic de novo fatty acid synthesis were measured in human cultured skin fibroblasts. Both activities in fibroblasts from normal controls and patients with adrenoleukodystrophy (ALD) were compared and slight but a significant increase of elongation activities in ALD fibroblasts was demonstrated. On the other hand, there were no significant differences in the fatty acid synthetase activities. In this study, we measured microsomal fatty acid elongation activities in the presence of N-ethylmaleimide, which completely inhibited the activity of contaminating fatty acid synthetase and also the degradation of fatty acids, and made accurate determination of the elongation activities possible.

Catalytic and structural properties of the dihydrolipoyl transacylase component of bovine branched-chain alpha-keto acid dehydrogenase.

Branched-chain alpha-keto acid dehydrogenase is a multienzyme complex consisting of three catalytic components, i.e. branched-chain alpha-keto acid decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3). In this report the E2 component of highly purified branched-chain alpha-keto acid dehydrogenase from bovine kidney and liver was characterized with an independent radiochemical assay for this component. The assay uses the model reaction: R-14CO-S-CoA + Lip-(SH)2 in equilibrium R-14CO-S-Lip-SH + CoA-SH, which is similar to that catalyzed by the transacetylase component of the pyruvate dehydrogenase complex. In this reaction, exogenous dihydrolipoamide substitutes for the protein (E2)-bound dihydrolipoyl moiety, and [1-14C]acyl-CoA synthesized enzymatically is the acyl-CoA substrate. The thioester structure of the reaction product, S-acyldihydrolipoamide, was identified by mass spectrometry, its characteristic absorption at 232-245 nm and by formation of hydroxamate with hydroxylamine. Rates of the E2-catalyzed transacylation reaction with various [1-14C]acyl-CoAs are in the order of [1-14C]isobutyryl-CoA greater than [1-14C] isovaleryl-CoA greater than [1-14C]acetyl-CoA. The activity with acetyl-CoA is 15% of that with isobutyryl-CoA. The E2 activity is strongly inhibited by arsenite. Modification of the covalently bound lipoyl moiety through reductive acylation in the presence of N-ethylmaleimide is without effect on the transacylation reaction. These data, along with results of initial velocity and product inhibition suggest that the model reaction proceeds via a random Bi Bi mechanism. Limited proteolysis of purified bovine liver branched-chain alpha-keto acid dehydrogenase with trypsin results in complete loss of the overall activity catalyzed by the complex. Nonetheless the activity of the E2 component is not affected. The tryptic digestion cleaves E2 subunits (Mr = 52,600) into a major fragment of Mr = 25,700. By contrast, E1 alpha and E1 beta subunits of the complex are relatively resistant to proteolysis with trypsin. The results indicate that structural properties of the E2 component of branched-chain alpha-keto acid dehydrogenase are similar but not identical to those of the transacetylase component of the pyruvate dehydrogenase complex.