Quantitation of Human Metallothionein Isoforms in Cells, Tissues, and Cerebrospinal Fluid by Mass Spectrometry.

Metallothioneins (MTs) are a family of small, highly conserved, cysteine-rich metal-binding proteins that are important for zinc and copper homeostasis, protection against oxidative stress, and buffering against toxic heavy metals. Individual human MT isoforms are candidate biomarkers for heavy metal toxicity, and selected cancers and neurodegenerative diseases. The similar antigenicity of human MT-1 and MT-2 isoforms precludes development of antibody-based assays for their individual quantitation. Metal-based MT quantitation methods do not directly measure MT isoforms. A bottom-up mass spectrometry-based approach solves these problems by exploiting the unique masses and chromatographic properties of the acetylated N-terminal tryptic peptides of MT isoforms. These unusually hydrophilic 20- to 21-residue peptides contain five invariant cysteines. Strong cation exchange chromatography separates them from bulk internal tryptic peptides. Reversed-phase chromatography further separates them from more hydrophobic peptides of similar mass. Absolute quantitation is obtained by adding MT peptide standards alkylated with 15N-iodoacetamide to biological samples alkylated with 14N-iodoacetamide. Accurate quantitation is enhanced by dimethyl sulfide treatment to reverse oxidation of the N-terminal methionine. Originally optimized for measuring MT isoforms in cell lines, the method has been adapted to quantify MT isoforms in brain tissue and cerebrospinal fluid. The method can also be adapted for relative quantitation of MT isoforms between matched biological samples. It cannot be used to measure human MT-4 because of an arginine at position 4. Except for this type of limitation, the method is applicable to MT quantitation in many other species.

Resonant mirror biosensor analysis of type Ialpha cAMP-dependent protein kinase B domain--cyclic nucleotide interactions.

A resonant mirror biosensor was used to study cyclic nucleotide-receptor interactions. In particular, a novel method was developed to determine inhibition constants (Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide-binding properties of the wild-type isolated B domain of the cAMP-dependent protein kinase type Ialpha regulatory subunit and its Ala-334-Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP-saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X-100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild-type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor-determined kinetic constants. Biosensor-determined relative inhibition constants (Ki' = Ki(cAMP)/Ki(analog)) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published Ki' values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The Ki' values for the wild-type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide-receptor interactions.

Characterization of the ATP- and GTP-specific succinyl-CoA synthetases in pigeon. The enzymes incorporate the same alpha-subunit.

Two succinyl-CoA synthetases, one highly specific for GTP/GDP and the other for ATP/ADP, have been purified to homogeneity from pigeon liver and breast muscle. The two enzymes are differentially distributed in pigeon, with only the GTP-specific enzyme detected in liver and the ATP-specific enzyme in breast muscle. Based on assays in the direction of CoA formation, the ratios of GTP-specific to ATP-specific activities in kidney, brain, and heart are approximately 7, 1, and 0.1, respectively. Both enzymes have the characteristic alpha- and beta-subunits found in other succinyl-CoA synthetases. Studies of the alpha-subunit by electrophoresis, mass spectrometry, reversed-phase high performance liquid chromatography, and peptide mapping showed that it was the same in the two enzymes. Characterization of the beta-subunits by the same methods indicated that they were different, with the tryptic peptide maps providing evidence that the beta-subunits likely differ along their entire sequences. Because the two succinyl-CoA synthetases incorporate the same alpha-subunit, the determinants of nucleotide specificity must reside within the beta-subunit. Determination of the apparent Michaelis constants showed that the affinity of the GTP-specific enzyme for GDP is greater than that of the ATP-specific enzyme for ADP (7 versus 250 microM). Rather large differences in apparent Km values were also observed for succinate and phosphate.

Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules.

We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.

The compartmentation of nucleoside diphosphate kinase in mitochondria.

The compartmentation of nucleoside diphosphate kinase (NDPK) was studied in mitochondria isolated from heart and liver of rat, rabbit, and pigeon. Compartmentation was assessed by determining latencies of enzyme activities, fractionating mitochondria with digitonin, and treating mitochondria with trypsin in the presence and absence of digitonin. NDPK activity in pigeon liver mitochondria was five- and seven-fold higher than in rat and rabbit liver mitochondria. The ratios of NDPK activities in liver vs. heart mitochondria were about 15 for rat, 2 for rabbit, and more than 40 for pigeon. Nearly all NDPK in pigeon liver mitochondria is in the matrix space, but outside the matrix in rat and rabbit liver mitochondria. Most NDPK in pigeon heart mitochondria was located outside the matrix while a significant fraction may be in the matrix of rat and rabbit heart mitochondria. These results are discussed relative to the assumed role that mitochondrial NDPK transfers the phosphoryl group of GTP produced in the Krebs cycle to the adenine nucleotide pool.

High-performance liquid chromatography-based assays of enzyme activities.

Interest in using HPLC to assay enzymatic reactions continues to grow as evidenced by the more than 100 papers published during the early 1990s. HPLC can be used for any enzymatic assay that requires separation of substrates and products before quantifying the extent of the reaction. The popularity of HPLC-based assays is due to several reasons: (1) HPLC offers unsurpassed precision, specificity, sensitivity, and reproducibility. (2) Powerful microcomputers and user-friendly software automate the running of samples and collection and processing of data. (3) Current columns, especially C18 packings, separate a very wide variety of samples, and (4) A variety of on-line detectors provide a means to detect virtually any compound. This review surveys recent papers on the development of HPLC-based assays for enzymes that degrade or otherwise modify macromolecules. Methods for assaying enzymes involved in metabolic pathways are also reviewed. Work by the authors in developing HPLC-based assays for mitochondrial enzymes that use GTP/GDP and other nucleotides that cannot be or are not easily assayed by enzyme-coupled assays is discussed. These enzymes include nucleoside diphosphate kinase, succinate thiokinase, and GTP-AMP phosphotransferase. The assays are suitable for determining the submitochondrial compartmentation of enzyme activities. Finally, current and anticipated trends in HPLC technology, including new column packings and the trend toward smaller columns that give faster separations, are reviewed in relation to enzyme assays.

The direct assay of kinases and acyl-CoA synthetases by HPLC: application to nucleoside diphosphate kinase and succinyl-CoA synthetase.

A general procedure is described for developing and validating HPLC-based assays for nucleotide-utilizing enzymes. The method is comparable in sensitivity to spectrophotometric, enzyme-coupled assays and is applicable to any enzymatic reaction involving coenzyme A and its esters, or the common nucleoside tri-, di-, and/or monophosphates. Nucleotide products of the enzymatic reactions are directly quantitated from their uv absorbances. A typical HPLC-based assay involves running an enzymatic reaction for a fixed time, stopping the reaction by adding acid, and using HPLC to quantitate the amount(s) of nucleotide reactant(s) converted to product(s). Nucleotides containing the same base can be separated under isocratic conditions using a 5-microns C-18, reversed-phase column, and a mobile phase at pH 5 containing 100 mM potassium phosphate, 20 mM acetate, 5 mM tetrabutylammonium ion as an ion-pairing agent, and sufficient acetonitrile to keep the run time within 10 min. Separation of nucleotides containing different bases can usually be accomplished under isocratic conditions by the proper choice of the pH within the range of 3 to 7. A microcomputer is used to control all system hardware and to automate collection and processing of chromatographic data. Application of the general method to development of assays for nucleoside diphosphate kinase and succinyl-CoA synthetase is described. The assay for nucleoside diphosphate kinase quantitates UTP formed from UDP and ATP as substrates. The assay for succinyl-CoA synthetase measures succinyl-CoA after HPLC separation of reactants and products at pH 4 where succinyl-CoA is stable to chemical hydrolysis.

Factors affecting the manganese and iron activation of the phosphoenolpyruvate carboxykinase isozymes from rabbit.

Timed assays in which GTP and GDP were separated and quantitated by HPLC were developed and used to study the metal activation of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase purified from rabbit liver. These assays allowed both directions of catalysis to be studied under similar conditions and in the absence of coupling enzymes. The mitochondrial enzyme is rapidly inactivated by preincubation with Fe2+, as had been shown previously for the cytosolic isozyme. The greatest activation by Fe2+ was obtained by adding micromolar Fe2+ immediately after enzyme to form the complete assay mixture that also contained millimolar Mg2+. In the direction of synthesis of OAA from Pep, the K0.5 values for Mn2+ and Fe2+ were in the 3-7 microM range when a nonchelating buffer, Hepes, was used. The buffer used strongly affected activation by Fe2+ at pH 7.4; activation was eliminated in the case of phosphate and K0.5 increased several-fold over that obtained with Hepes when imidazole was used. In non-chelating buffer, the pH optimum was near 7.4 for both isozymes and for both directions of catalysis. However, the near optimal pH range extended below 7.4 for the direction of oxaloacetate synthesis while the range was above 7.4 for Pep synthesis. In the direction of oxaloacetate synthesis: (1) Both isozymes required the presence of micromolar Mn2+ or Fe2+ in addition to millimolar Mg2+ in order to shown significant activity. (2) Fe2+ was as effective an activator as Mn2+ at pH 7 and below. In the direction of Pep synthesis: (1) Micromolar Mn2+ was a much better activator than Fe2+ at the higher pH values needed for optimal activity in this direction. (2) With increasing pH, decreasing activation was obtained with Fe2+ while the activity supported by Mg2+ alone increased. The results demonstrate the potential for regulation of either isozyme of Pep carboxykinase by the availability of iron or manganese.