Gary A. Radke

Lipoyl Domain-based Mechanism for the Integrated Feedback Control of the Pyruvate Dehydrogenase Complex by Enhancement of Pyruvate Dehydrogenase Kinase Activity

To conserve carbohydrate reserves, the reaction of the pyruvate dehydrogenase complex (PDC) must be down-regulated when the citric acid cycle is provided sufficient acetyl-CoA. PDC activity is reduced primarily through increased phosphorylation of its pyruvate dehydrogenase (E1) component due to E1 kinase activity being markedly enhanced by elevated intramitochondrial NADH:NAD+ and acetyl-CoA:CoA ratios. A mechanism is evaluated in which enhanced kinase activity is facilitated by the build-up of the reduced and acetylated forms of the lipoyl moieties of the dihydrolipoyl acetyltransferase (E2) component through using NADH and acetyl-CoA in the reverse of the downstream reactions of the complex. Using a peptide substrate, kinase activity was stimulated by these products, ruling out the possibility kinase activity is increased due to changes in the reaction state of its substrate, E1 (thiamin pyrophosphate). Each E2 subunit contains two lipoyl domains, an NH2-terminal (L1) and the inward lipoyl domain (L2), which were individually produced in fully lipoylated forms by recombinant techniques. Although reduction and acetylation of the L1 domain or free lipoamide increased kinase activity, those modifications of the lipoate of the kinase-binding L2 domain gave much greater enhancements of kinase activity. The large stimulation of the kinase generated by acetyl-CoA only occurred upon addition of the transacetylase-catalyzing (lipoyl domain-free) inner core portion of E2 plus a reduced lipoate source, affirming that acetylation of this prosthetic group is an essential mechanistic step for acetyl-CoA enhancing kinase activity. Similarly, the lesser stimulation of kinase activity by just NADH required a lipoate source, supporting the need for lipoate reduction by E3 catalysis. Complete enzymatic delipoylation of PDC, the E2-kinase subcomplex, or recombinant L2 abolished the stimulatory effects of NADH and acetyl-CoA. Retention of a small portion of PDC lipoates lowered kinase activity but allowed stimulation of this residual kinase activity by these products. Reintroduction of lipoyl moieties, using lipoyl protein ligase, restored the capacity of the E2 core to support high kinase activity along with stimulation of that activity up to 3-fold by NADH and acetyl-CoA. As suggested by those results, the enhancement of kinase activity is very responsive to reductive acetylation with a half-maximal stimulation achieved with ∼20% of free L2 acetylated and, from an analysis of previous results, with acetylation of only 3-6 of the 60 L2 domains in intact PDC. Based on these findings, we suggest that kinase stimulation results from modification of the lipoate of an L2 domain that becomes specifically engaged in binding the kinase. In conclusion, kinase activity is attenuated through a substantial range in response to modest changes in the proportion of oxidized, reduced, and acetylated lipoyl moieties of the L2 domain of E2 produced by fluctuations in the NADH:NAD+ and acetyl-CoA:CoA ratios as translated by the rapid and reversible E3 and E2 reactions.

Aqueous Solubilization of Transmembrane Peptide Sequences with Retention of Membrane Insertion and Function

We recently reported that the peptide C-K4-M2GlyR mimics the action of chloride channels when incorporated into the apical membrane of cultured renal epithelial monolayers. C-K4-M2GlyR is one of a series of peptides that were prepared by the addition of lysine residues to the N- or C-terminus of the M2 transmembrane sequence of the brain glycine receptor. This study addresses how such modifications affect physical properties such as aqueous solubility, aggregation, and secondary structure, as well as the ability of the modified peptides to form channels in epithelial monolayers. A graded improvement in solubility with a concomitant decrease in aggregation in aqueous media was observed for the M2GlyR transmembrane sequences. Increases in short-circuit current (I(SC)) of epithelial monolayers were observed after treatment with some but not all of the peptides. The bioactivity was higher for the more soluble, less aggregated M2GlyR peptides. As described in our previous communication, sensitivity of channel activity to diphenylamine-2-carboxylate, a chloride channel blocker, and bumetanide, an inhibitor of the Na/K/2Cl cotransporter, was used to assess changes in chloride selectivity for the different assembled channel-forming peptides. The unmodified M2GlyR sequence and the modified peptides with less positive charge are more sensitive to these agents than are the more highly charged forms. This study shows that relatively insoluble transmembrane sequences can be modified such that they are easier to purify and deliver in the absence of organic solvents with retention of membrane association, insertion, and assembly.

Role of protein X in the function of the mammalian pyruvate dehydrogenase complex

Two lipoyl-bearing subunits--the dihydrolipoyl transacetylase and protein X--form the core of the mammalian pyruvate dehydrogenase complex. Selective removal of the lipoyl domain of protein X results in loss in the activity of the complex with a relationship suggesting the involvement of the lipoyl domain of protein X in a key but not rate limiting step. The dihydrolipoyl dehydrogenase component markedly reduces both the cleavage of protein X and the loss in activity. Using a microplate binding assay, we demonstrate that the lipoyl domain of protein X and the transacetylase component contribute to the binding of the dihydrolipoyl dehydrogenase component. These roles of protein X in the catalytic function and organization of the complex require new reactions and afford an explanation for the unusual stoichiometry of dihydrolipoyl dehydrogenase dimers in the complex.

Measurement of protein using flow injection analysis with bicinchoninic acid

We have used the bicinchoninic acid reagent developed by Pierce Chemical Co. to measure proteins in a simple flow injection analyzer. The sensitivity is comparable to that of the Lowry method and no pipetting of reagents is needed. Results are obtained in less than 1 min and samples may be run at a rate of 60/h. The response is linear over a range of protein concentration (0-10 micrograms) and sample size (5-20 microliters) convenient for most analytical requirements. A peristaltic pump, a controlled-temperature water bath, and a spectrophotometer with flow cuvette are the only special apparatus required.

On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS.

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3...). The high-energy collision-induced dissociation (heCID) spectra of labeled KS oligosaccharides displayed fragments of compounds similar to those observed with laser-induced dissociation (LID) analysis, suggesting that both heCID and LID fragmentations can be used to analyze KS oligosaccharides. Moreover, fragmentation analysis of all labeled KS oligosaccharides was performed by MALDI-TOF/TOF-MS. With LID mode, sodium adducts showed fragmentation of glycosidic linkages with mainly Y/B/C ions, as well as various cross-ring cleavages providing exact information for the positions of sulfate groups along the KS oligosaccharide chains. This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples.

Synthesis of N-t-Boc-4-S-t-Butyl-L-thiophenylalanine via Palladium Catalyzed Cross-Coupling Reaction of N-t-Boc-4-Iodo-L-phenylalanine with t-Butylthiol or Sodium t-Butylthiolate

Abstract Cross-coupling reaction of N-t-Boc-4-iodo-L-phenylalanine with t-BuSH proceeded efficiently in the presence of Pd2dba3.CHCl3/DPPF as the catalyst system. However, Pd2dba3.CHCl3/DPPB was found to be more effective for coupling with t-BuSNa.

Arcelins from an Indian Wild Pulse, Lablab purpureus, and Insecticidal Activity in Storage Pests

A partially purified protein fraction was isolated from seed flour of the Indian wild bean, Lablab purpureus, by ion exchange and size-exclusion chromatographies. Partially purified L. purpureus proteins had hemagglutination and glycoslyation properties similar to those of lectins or lectin-like proteins from other pulses. Data obtained from two-dimensional gel electrophoresis, MALDI-TOF, and MALDI-TOF/TOF and N-terminal protein sequencing of the isolated polypeptides from L. purpureus demonstrated that the extract contained proteins similar to isoforms of arcelins 3 and 4 and pathogenesis-related protein 1 (PvPR1) of Phaseolus vulgaris. L. purpureus proteins were resistant to degradation by the commercial enzymes trypsin and chymotrypsin and were moderately resistant to pepsin, but were readily hydrolyzed to smaller peptides by papain. Insect feeding bioassays of the extract with the storage pests Rhyzopertha dominica and Oryzaephilus surinamensis, internal and external feeders of grain, respectively, demonstrated that L. purpureus proteins at 2% in the diet resulted in retarded development. However, a 5% dose of the L. purpureus fraction resulted in complete mortality of all larvae in both species. This study has demonstrated that proteins in the partially purified L. purpureus extract have the potential to control storage pests in cereals transformed with L. purpureus defense-related genes, but the need for more studies regarding efficacy and safety is discussed.

Additional binding sites for the pyruvate dehydrogenase kinase but not for protein X in the assembled core of the mammalian pyruvate dehydrogenase complex: Binding region for the kinase

A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.

Lipoyl-Containing Components of the Pyruvate Dehydrogenase Complex: Roles in Modulating and Anchoring the PDH Kinase and the PDH Phosphatasea

The activity of the pyruvate dehydrogenase, (PDH,) kinase lowers the fraction of the pyruvate dehydrogenase complex that is present in the active form through phosphorylating and concomitantly inactivating the pyruvate dehydrogenase component (PDH, or El ). The pyruvate dehydrogenaseb (PDH,) phosphatase dephosphorylates the a subunit of the E l component and thereby activates the pyruvate dehydrogenase complex. In this paper, we will describe the role of the lipoyl-containing componentsthe dihydrolipoyl transacetylase and protein X-in the binding and in the regulation of the kinase and the phosphatase purified from bovine kidney or heart.

Chemically coupled spectrophotometric assays based on flow injection analysis: Determination of nitrogenase by assays for creatine, ammonia, hydrazine, phosphate, and dithionite

Micromethods of direct chemical coupling have been developed for several different enzyme reactions, using the principles of flow injection analysis. Samples of 1-25 microliters are injected into a flowing stream of color-forming reagents and the peak of color change is measured after about 1 min. Alternatively, continuous slow infusion of a reacting system (5-100 microliters/min) gives a continuous change of color which can be monitored to derive enzyme reaction rates. These techniques are highly sensitive, requiring a few nanomoles of the substance being detected. Phosphate, ammonia, dithionite, creatine, and hydrazine have been measured. Consumption of reagents is less than 75 ml per hour; typical sample throughout is 30-40 samples per hour by the injection method, and 5 samples per hour by continuous infusion. The procedure has been applied to nitrogenase, continuously monitoring creatine produced from creatine phosphate by creatine kinase which is used to supply a constant level of ATP for nitrogenase. In this way nitrogenase activity can be determined over a wide range of enzyme concentrations. Production of inorganic phosphate directly from ATP, by injection of formaldehyde-quenched samples, was used when coupling to creatine kinase was not possible. Both injection of aliquots and continuous infusion were used for detection of hydrazine during nitrogenase reduction of azide, and the injection method has been used for ammonia assay during dinitrogen reduction. Dithionite oxidation was measured directly from decolorization of iodine, after trapping both dithionite and bisulfite with formaldehyde.