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Parkinson's disease (PD), characterized by dopaminergic neuronal loss, is attributed to oxidative stress, diminished glutathione (GSH) levels, mitochondrial dysfunction, and protein aggregation. Treatment of PD involves chronic administration of Levodopa (l-DOPA) which is a pro-oxidant and may disrupt sulfhydryl homeostasis. The goal of these studies is to elucidate the effects of l-DOPA on thiol homeostasis in a model akin to PD, i.e., immortalized dopaminergic neurons (SHSY5Y cells) with diminished GSH content. These neurons exhibit hypersensitivity to l-DOPA-induced cell death, which is attributable to concomitant inhibition of the intracellular thiol disulfide oxidoreductase enzymes. Glutaredoxin (Grx) was deactivated in a dose-dependent fashion, but its content was unaffected. Glutathione disulfide (GSSG) reductase (GR) activity was not altered. Selective knockdown of Grx resulted in an increased level of apoptosis, documenting the role of the Grx system in neuronal survival. l-DOPA treatments also led to decreased activities of thioredoxin (Trx) and thioredoxin reductase (TR), concomitant with diminution of their cellular contents. Selective chemical inhibition of TR activity led to an increased level of apoptosis, documenting the Trx system's contribution to neuronal viability. To investigate the mechanism of inhibition at the molecular level, we treated the each isolated enzyme with oxidized l-DOPA. GR, Trx, and TR activities were little affected. However, Grx was inactivated in a time- and concentration-dependent fashion indicative of irreversible adduction of dopaquinone to its nucleophilic active-site Cys-22, consistent with the intracellular loss of Grx activity but not Grx protein content after l-DOPA treatment. Overall l-DOPA is shown to impair the collaborative contributions of the Grx and Trx systems to neuron survival.

As resistance determinants, KPC β-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type β-lactamases are often identified as resistant to standard β-lactam-β-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to β-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios ( k cat / k inact ratios, where k inact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp 2 -hybridized C 3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: K m of penem 1, 0.06 ± 0.01 μM, and K m of penem 2, 0.006 ± 0.001 μM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios ( k cat / k inact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor ( k inact / K m = 6.5 ± 0.6 μM −1 s −1 ). We conclude that KPC-2 is unique among class A β-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type β-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current β-lactamase inhibitors.

In an effort to devise strategies for overcoming bacterial β-lactamases, we studied LN-1-255, a 6-alkylidene-2′-substituted penicillin sulfone inhibitor. By possessing a catecholic functionality that resembles a natural bacterial siderophore, LN-1-255 is unique among β-lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing blaSHV extended-spectrum and inhibitor-resistant β-lactamases than an equivalent amount of tazobactam and piperacillin. In addition, LN-1-255 significantly enhanced the activity of ceftazidime and cefpirome against extended-spectrum cephalosporin and Sme-1 containing carbapenem-resistant clinical strains. LN-1-255 inhibited SHV-1 and SHV-2 β-lactamases with nm affinity (KI = 110 ± 10 and 100 ± 10 nm, respectively). When LN-1-255 inactivated SHV β-lactamases, a single intermediate was detected by mass spectrometry. The crystal structure of LN-1-255 in complex with SHV-1 was determined at 1.55A resolution. Interestingly, this novel inhibitor forms a bicyclic aromatic intermediate with its carbonyl oxygen pointing out of the oxyanion hole and forming hydrogen bonds with Lys-234 and Ser-130 in the active site. Electron density for the “tail” of LN-1-255 is less ordered and modeled in two conformations. Both conformations have the LN-1-255 carboxyl group interacting with Arg-244, yet the remaining tails of the two conformations diverge. The observed presence of the bicyclic aromatic intermediate with its carbonyl oxygen positioned outside of the oxyanion hole provides a rationale for the stability of this inhibitory intermediate. The 2′-substituted penicillin sulfone, LN-1-255, is proving to be an important lead compound for novel β-lactamase inhibitor design.

For the proteomic study of mitochondrial membranes, documented high quality mitochondrial preparations are a necessity to ensure proper localization. Despite the state-of-the-art technologies currently in use, there is no single technique that can be used for all studies of mitochondrial membrane proteins. Herein, we use examples to highlight solubilization techniques, different chromatographic methods, and developments in gel electrophoresis for proteomic analysis of mitochondrial membrane proteins. Blue-native gel electrophoresis has been successful not only for dissection of the inner membrane oxidative phosphorylation system, but also for the components of the outer membrane such as those involved in protein import. Identification of PTMs such as phosphorylation, acetylation, and nitration of mitochondrial membrane proteins has been greatly improved by the use of affinity techniques. However, understanding of the biological effect of these modifications is an area for further exploration. The rapid development of proteomic methods for both identification and quantitation, especially for modifications, will greatly impact the understanding of the mitochondrial membrane proteome.

Measurement of complex III activity is critical to the diagnosis of human mitochondrial disease and the study of mitochondrial pathobiology. Activity is measured as the maximal rate of antimycin A-sensitive reduction of exogenous cytochrome c by detergent-solubilized mitochondria. Complex III activity exhibited an unexpected variation based upon the commercial source of cytochrome c owing to an increase in the antimycin A-insensitive background reduction of cytochrome c and variable increases in total activity. Analysis of cytochrome c (producing a high-background) by fast protein liquid chromatography yielded a contaminant peak containing a lipid extractable component with redox spectra and mass spectroscopy fragmentation suggestive of a quinol. Measurement of inhibitor-sensitive rates are critical for the accurate and reproducible measurement of complex III activity and serve as a key quality control to screen for non-enzymatic reactions that obscure complex III activity.

The identification of post-translational modifications is difficult especially for hydrophobic membrane proteins. Here we present the identification of several types of protein modifications on membrane proteins isolated from mitochondrial outer membranes. We show, in vivo, that the mature rat liver mitochondrial carnitine palmitoyltransferase-I enzyme is N-terminally acetylated, phosphorylated on two threonine residues, and nitrated on two tyrosine residues. We show that long chain acyl-CoA synthetase 1 is acetylated at both the N-terminal end and at a lysine residue and tyrosine residues are found to be phosphorylated and nitrated. For the three voltage-dependent anion channel isoforms present in the mitochondria, the N-terminal regions of the protein were determined and sites of phosphorylation were identified. These novel findings raise questions about regulatory aspects of carnitine palmitoyltransferase-I, long chain acyl-CoA synthetase and voltage dependent anion channel and further studies should advance our understanding about regulation of mitochondrial fatty acid oxidation in general and these three proteins in specific.

Inhibitor-resistant class A β-lactamases are an emerging threat to the use of β-lactam/β-lactamase inhibitor combinations (e.g. amoxicillin/clavulanate) in the treatment of serious bacterial infections. In the TEM family of Class A β-lactamases, single amino acid substitutions at Arg-244 confer resistance to clavulanate inactivation. To understand the amino acid sequence requirements in class A β-lactamases that confer resistance to clavulanate, we performed site-saturation mutagenesis of Arg-244 in SHV-1, a related class A β-lactamase found in Klebsiella pneumoniae. Twelve SHV enzymes with amino acid substitutions at Arg-244 resulted in significant increases in minimal inhibitory concentrations to ampicillin/clavulanate when expressed in Escherichia coli. Kinetic analyses of SHV-1, R244S, R244Q, R244L, and R244E β-lactamases revealed that the main determinant of clavulanate resistance was reduced inhibitor affinity. In contrast to studies in the highly similar TEM enzyme, we observed increases in clavulanate kinact for all mutants. Electrospray ionization mass spec-trometry of clavulanate inhibited SHV-1 and R244S showed nearly identical mass adducts, arguing against a difference in the inactivation mechanism. Testing a wide range of substrates with C3-4 carboxylates in different stereochemical orientations, we observed impaired affinity for all substrates among inhibitor resistant variants. Lastly, we synthesized two boronic acid transition state analogs that mimic cephalothin and found substitutions at Arg-244 markedly affect both the affinity and kinetics of binding to the chiral, deacylation transition state inhibitor. These data define a role for Arg-244 in substrate and inhibitor binding in the SHV β-lactamase.

Objective Despite the growing body of evidence showing adverse health effects from inhalation exposure to the trichothecene-producing mold Stachybotrys chartarum, controversy remains. Currently, there are no reliable assays suitable for clinical diagnosis of exposure. We hypothesized that satratoxin G (SG)–albumin adducts may serve as biomarkers of exposure to this fungus. Design We studied the formation of adducts of SG with serum albumin in vitro using Western blots and mass spectrometry (MS) and searched for similar adducts formed in vivo using human and animal serum. Results Samples of purified human serum albumin that had been incubated with increasing concentrations of SG showed concentration-dependent albumin bands in Western blots developed with anti-SG antibodies. MS analysis found that as many as 10 toxin molecules can be bound in vitro to one albumin molecule. The sequencing of albumin-adduct tryptic peptides and the analysis of pronase/aminopeptidase digests demonstrated that lysyl, cysteinyl, and histidyl residues are involved in the formation of these adducts. Serum samples from three patients with documented exposure to S. chartarum similarly revealed lysine–, cysteine–, and histidine–SG adducts after exhaustive digestion, affinity column enrichment, and MS analysis. These adducts were also found in the sera from rats exposed to the spores of S. chartarum in contrast to control human subjects and control animals. Conclusions These data document the occurrence of SG–albumin adducts in both in vitro experiments and in vivo human and animal exposures to S. chartarum. Relevance to clinical practice SG–amino acid adducts may serve as reliable dosimeter biomarkers for detection of exposure to S. chartarum.

Hepatic carnitine palmitoyltransferase-I (CPT-IL) isolated from mitochondrial outer membranes obtained in the presence of protein phosphatase inhibitors is readily recognized by phosphoamino acid antibodies. Mass spectrometric analysis of CPT-IL tryptic digests revealed the presence of three phosphopeptides including one with a protein kinase CKII (CKII) consensus site. Incubation of dephosphorylated outer membranes with protein kinases and [γ-32P]ATP resulted in radiolabeling of CPT-I only by CKII. Using mass spectrometry, only one region of phosphorylation was detected in CPT-I isolated from CKII-treated mitochondria. The sequence of the peptide and position of phosphorylated amino acids have been determined unequivocally as FpSSPETDpSHRFGK (residues 740-752). Furthermore, incubation of dephosphorylated outer membranes with CKII and unlabeled ATP led to increased catalytic activity and rendered malonyl-CoA inhibition of CPT-I from competitive to uncompetitive. These observations identify a new mechanism for regulation of hepatic CPT-I by phosphorylation.

Molecular complexes such as double-stranded oligonucleotides contain non-covalent bonds that are difficult to maintain in the MALDI experiment. Quantifiers are introduced in order to evaluate, summarize, and compare spectra from experiments in which additives are used to stabilize duplex oligonucleotides. Compounds known to complex with and stabilize duplex molecules can be useful as additives in MALDI. Spermine and methylene blue, present at concentrations similar to the matrix, are detected, bound to the duplex. When peptides are used as additives, the duplex is stabilized when the peptide is present at an amount less than that of the duplex.

With the development of matrix additives, the MALDI-MS analysis of oligonucleotides has improved greatly. When the monosaccharide fucose is combined with the matrix, the homogeneity of the MALDI target, signal strength, and signal duration are increased. The sensitivity of the MALDI experiment increases, allowing for improved detection of components in a complex mixture of oligonucleotides, such as that encountered in sequencing experiments. The addition of fucose to the matrix also causes a reduction in the extent of fragmentation of oligonucleotides.

Currently, CTX-M β-lactamases are among the most prevalent and most heterogeneous extended-spectrum β-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by β-lactamase inhibitors. However, it is unknown if the pathway to inhibition by β-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV β-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 β-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. K i values ranged from 1.6 ± 0.3 μM (mean ± standard error) for tazobactam to 0.02 ± 0.01 μM for penem 1. Before and after tryptic digestion of the CTX-M-9 β-lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 ± 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 β-lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C 3 carboxylate of inhibitors and that the localization of this positive charge to a “region of the active site” rather than a specific residue represents an important evolutionary strategy used by β-lactamases.

Class D {beta}-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial {beta}-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel {beta}-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2{prime}-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important {beta}-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 {beta}-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC{sub 50} values against OXA-24 and two OXA-24 {beta}-lactamase variants ranged from 10 {+-} 1 (4 vs WT) to 338 {+-} 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K{sub i} (500 {+-} 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k{sub inact}/K{sub i} = 0.21 {+-} 0.02 {micro}M{sup -1}s{sup -1}). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 {angstrom}) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanionmore » hole. These data provide the first structural evidence that 6-alkylidene-2{prime}-substituted penicillin sulfones are effective mechanism-based inactivators of class D {beta}-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D {beta}-lactamases is proposed.« less

The need to develop beta-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C(3)/C(4) carboxylate of beta-lactams showed the lowest K(i) (11 +/- 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla(ADC) from 16 to 4 microg/mL and from 8 to 1 microg/mL, respectively. On the other hand, each carbapenem exhibited a K(i) of

Class D beta-lactamases hydrolyze beta-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate and subsequently employ water to deacylate the beta-lactam and release product. Class D beta-lactamases are carboxylated on the epsilon-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 beta-lactamase, a monomeric class D enzyme, significantly disrupt catalytic turnover. Substitution of glycine for the nucleophilic serine (S67G) results in an enzyme that can still bind substrate but is unable to form a covalent acyl-enzyme intermediate. Substitution of the carboxylated lysine (K70), on the other hand, results in enzyme that can be acylated by substrate but is impaired with respect to deacylation. We employed the fluorescent penicillin BOCILLIN FL to show that three different substitutions for K70 (alanine, aspartate, and glutamate) lead to the accumulation of significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL deacylation rates (t(1/2)) vary depending on the identity of the substituting residue, from approximately 60 min for K70A to undetectable deacylation for K70D. Tryptophan fluorescence spectroscopy was used to confirm that these results are applicable to natural (i.e., nonfluorescent) substrates. Deacylation by K70A, but not K70D or K70E, can be partially restored by the addition of short-chain carboxylic acid mimetics of the lysine carbamate. In conclusion, we establish the functional role of the carboxylated lysine in OXA-1 and highlight its specific role in acylation and deacylation.

In recent years, a wide variety of proteomic approaches using gel electrophoresis and mass spectrometry has been developed to detect post-translational modifications. Mitochondria are often a focus of these studies due to their important role in cellular function. Many of their crucial transport and oxidative-phosphorylation functions are performed by proteins residing in the inner and outer membranes of the mitochondria. Although proteomic technologies have greatly enhanced our understanding of regulation in cellular processes, analysis of membrane proteins has lagged behind that of soluble proteins. Herein, we present techniques to facilitate the detection of post-translational modifications of mitochondrial membrane proteins including the isolation of resident membranes as well as electrophoretic and immunological-based methods for identification of post-translational modifications.

Protein S-glutathionylation is a reversible redox-dependent post-translational modification. Many cellular functions and signal transduction pathways involve proteins whose cysteine-dependent activities are modulated by glutathionylation. Glutaredoxin (Grx1) plays a key role in such regulation because it is a specific and efficient catalyst of deglutathionylation. We recently reported an increase in Grx1 in retinae of diabetic rats and in rat retinal Müller glial cells (rMC-1) cultured in high glucose. This up-regulation of Grx1 was concomitant with NFκB activation and induction of intercellular adhesion molecule-1 (ICAM-1). This proinflammatory response was replicated by adenoviral-directed up-regulation of Grx1 in cells in normal glucose. The site of regulation of NFκB was localized to the cytoplasm, where IκB kinase (IKK) is a master regulator of NFκB activation. In the current study, inhibition of IKK activity abrogated the increase in ICAM-1 induced by high glucose or by adenoviral-directed up-regulation of Grx1. Conditioned medium from the Müller cells overexpressing Grx1 was added to fresh cultures of Müller or endothelial cells and elicited increases in the Grx1 and ICAM-1 proteins in these cells. These effects correlate with a novel finding that secretion of interleukin-6 was elevated in the cultures of Grx overexpressing cells. Also, pure interleukin-6 increased Grx1 and ICAM-1 in the rMC-1 cells. Thus, Grx1 appears to play an important role in both autocrine and paracrine proinflammatory responses. Furthermore, IKKβ isolated from Müller cells in normal glucose medium was found to be glutathionylated on Cys-179. Hence Grx-mediated activation of IKK via deglutathionylation may play a central role in diabetic complications in vivo where Grx1 is increased.

Inhibitor-resistant class A beta-lactamases of the TEM and SHV families that arise by single amino acid substitutions are a significant threat to the efficacy of beta-lactam/beta-lactamase inhibitor combinations. To better understand the basis of the inhibitor-resistant phenotype in SHV, we performed mutagenesis to examine the role of a second-shell residue, Asn276. Of the 19 variants expressed in Escherichia coli, only the Asn276Asp enzyme demonstrated reduced susceptibility to ampicillin/clavulanate (MIC increased from 50/2 --50/8 microg/mL) while maintaining high-level resistance to ampicillin (MIC = 8192 microg/mL). Steady-state kinetic analyses of Asn276Asp revealed slightly diminished k(cat)/K(m) for all substrates tested. In contrast, we observed a 5-fold increase in K(i) for clavulanate (7.4 +/- 0.9 microM for Asn276Asp vs 1.4 +/- 0.2 microM for SHV-1) and a 40% reduction in k(inact)/K(I) (0.013 +/- 0.002 microM(-1 )s(-1) for Asn276Asp vs 0.021 +/- 0.004 microM(-1) s(-1) for SHV-1). Timed electrospray ionization mass spectrometry of clavulanate-inhibited SHV-1 and SHV Asn276Asp showed nearly identical mass adducts, arguing for a similar pathway of inactivation. Molecular modeling shows that novel electrostatic interactions are formed between Arg244Neta2 and both 276AspOdelta1 and Odelta2; these new forces restrict the spatial position of Arg244, a residue important in the recognition of the C(3)/C(4) carboxylate of beta-lactam substrates and inhibitors. Testing the functional consequences of this interaction, we noted considerable free energy costs (+DeltaDeltaG) for substrates and inhibitors. A rigid carbapenem (meropenem) was most affected by the Asn276Asp substitution (46-fold increase in K(i) vs SHV-1). We conclude that residue 276 is an important second-shell residue in class A beta-lactamase-mediated resistance to substrates and inhibitors, and only Asn is able to precisely modulate the conformational flexibility of Arg244 required for successful evolution in nature.

In recent years, a wide variety of proteomic approaches using gel electrophoresis and mass spectrometry has been developed to detect post-translational modifications. Mitochondria are often a focus of these studies due to their important role in cellular function. Many of their crucial transport and oxidative-phosphorylation functions are performed by proteins residing in the inner and outer membranes of the mitochondria. Although proteomic technologies have greatly enhanced our understanding of regulation in cellular processes, analysis of membrane proteins has lagged behind that of soluble proteins. Herein, we present techniques to facilitate the detection of post-translational modifications of mitochondrial membrane proteins including the isolation of resident membranes as well as electrophoretic and immunological-based methods for identification of post-translational modifications.

The carnitine palmitoyltransferase-I (CPT-I) enzymes catalyze the regulated step in overall mitochondrial fatty acid oxidation. The liver and muscle isoforms are expressed in liver and skeletal muscle respectively with the isoforms exhibiting different kinetic properties and apparent molecular weight masses. In contrast, the heart expresses both isoforms at the mRNA level. However, for the expression of the liver isoform at the protein level only indirect evidence is available, such as tagging with radiolabeled CPT-I inhibitors followed by SDS-PAGE separation and kinetic analysis using inhibitors. The importance of fatty acid oxidation in the heart and the potential regulation via the liver isoform of CPT-I demands proof of the liver isoform in the heart. Using a proteomic approach in the present study we demonstrate that rat heart mitochondria (a) contain both the muscle and liver isoforms; (b) both proteins retain their C- and N-termini; (c) the N-terminal alanine residues are acetylated; (d) and in rat heart mitochondria the liver isoform is phosphorylated on tyrosine 281. By providing amino acid sequence information this is the first unequivocal demonstration that the liver isoform of CPT-I is expressed at the protein level in adult rat heart mitochondria and that the apparent smaller molecular size of the muscle isoform is not due to proteolytic truncation.

The partnering of a β-lactam with a β-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to β-lactam antibiotics mediated by serine β-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D β-lactamase that is resistant to inactivation by tazobactam. The K i of each penem inhibitor for OXA-1 was in the nM range ( K i of penem 1, 45 ± 8 nM; K i of penem 2, 12 ± 2 nM). The first-order rate constant for enzyme and inhibitor complex inactivation of penems 1 and 2 for OXA-1 β-lactamase were 0.13 ± 0.01 s −1 and 0.11 ± 0.01 s −1 , respectively. By using an inhibitor-to-enzyme ratio of 1:1, 100% inactivation was achieved in ≤900 s and the recovery of OXA-1 β-lactamase activity was not detected at 24 h. Covalent adducts of penems 1 and 2 (changes in molecular masses, +306 ± 3 and +321 ± 3 Da, respectively) were identified by electrospray ionization mass spectrometry (ESI-MS). After tryptic digestion of OXA-1 inactivated by penems 1 and 2, ESI-MS and matrix-assisted laser desorption ionization-time-of-flight MS identified the adducts of 306 ± 3 and 321 ± 3 Da attached to the peptide containing the active-site Ser67. The base hydrolysis of penem 2, monitored by serial 1 H nuclear magnetic resonance analysis, suggested that penem 2 formed a linear imine species that underwent 7-endo-trig cyclization to ultimately form a cyclic enamine, the 1,4-thiazepine derivative. Susceptibility testing demonstrated that the penem inhibitors at 4 mg/liter effectively restored susceptibility to piperacillin. Penem β-lactamase inhibitors which demonstrate high affinities and which form long-lived acyl intermediates may prove to be extremely useful against the broad range of inhibitor-resistant serine β-lactamases present in gram-negative bacteria.

Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 -->

Case Description—Acute pulmonary hemorrhage developed during isoflurane anesthesia in 2 Himalayan cats undergoing routine dental cleaning and prophylaxis. Clinical Findings—The cats were siblings and lived together. In both cats, results of pre-operative physical examinations and laboratory testing were unremarkable. Blood pressure and oxygen saturation were within reference ranges throughout the dental procedure. Approximately 15 to 20 minutes after administration of isoflurane was begun, frothy blood was noticed within the endotracheal tube. Blood was suctioned from the endotracheal tube, and the cats were allowed to recover from anesthesia. Treatment and Outcome—1 cat initially responded to supportive care but developed a second episode of spontaneous pulmonary hemorrhage approximately 30 hours later and died. The other cat responded to supportive care and was discharged after 4 days, but its condition deteriorated, and the cat died 10 days later. Subsequently, it was discovered that the home was severely contaminated with mold as a result of storm damage that had occurred approximately 7 months previously. Retrospective analysis of banked serum from the cats revealed satratoxin G, a biomarker for Stachybotrys chartarum, commonly referred to as “toxic black mold.” Clinical Relevance—Findings highlight the potential risk of acute pulmonary hemorrhage in animals living in an environment contaminated with mold following flood damage.

Inhibitor resistant class A beta-lactamases are an emerging threat to treatment of gram negative infections. Based on clinical isolates of inhibitor (clavulanate) resistant TEM enzymes with mutatio...

Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82-99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.

Hepatic carnitine palmitoyltransferase-I (CPT-IL) isolated from mitochondrial outer membranes obtained in the presence of protein phosphatase inhibitors is readily recognized by phosphoamino acid antibodies. Mass spectrometric analysis of CPT-IL tryptic digests revealed the presence of three phosphopeptides including one with a protein kinase CKII (CKII) consensus site. Incubation of dephosphorylated outer membranes with protein kinases and [gamma-32P]ATP resulted in radiolabeling of CPT-I only by CKII. Using mass spectrometry, only one region of phosphorylation was detected in CPT-I isolated from CKII-treated mitochondria. The sequence of the peptide and position of phosphorylated amino acids have been determined unequivocally as FpSSPETDpSHRFGK (residues 740-752). Furthermore, incubation of dephosphorylated outer membranes with CKII and unlabeled ATP led to increased catalytic activity and rendered malonyl-CoA inhibition of CPT-I from competitive to uncompetitive. These observations identify a new mechanism for regulation of hepatic CPT-I by phosphorylation.

The cytochrome c oxidase enzyme from Rhodobacter sphaeroides bacteria exists as a complex of four peptide subunits, two hemes and a variety of lipids and metal ions held together by non-covalent forces. Although the native enzyme functions as an associated unit, this complex usually dissociates during matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry analysis. Through the use of matrix additives such as sucrose, the complete complex and partial complexes can be stabilized in the MALDI-ToF experiment. The dissociation of the complex allows the detection of the components of the enzyme. The direct detection of associated lipids from an aqueous solution of the intact enzyme may eliminate the need for enzyme disruption and lipid extraction. The partial dissociation of multi-subunit enzymes in such experiments may allow the determination of subunit–subunit and subunit–lipid interactions.

5-Methoxysalicylic acid (MSA) is demonstrated to be a useful matrix for matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry of oligonucleotides, when desorption/ionization without fragmentation is desired. When MSA is combined with the additive spermine, the need for desalting is reduced. The MSA/spermine matrix yields linear TOF mass spectra with improved resolution, less fragmentation, and less intense alkali ion adduct peaks than those spectra obtained using 3-hydroxypicolinic acid and 6-aza-2-thiothymine with spermine or diammonium hydrogen citrate as additives. Instrumental conditions are discussed to improve the spectral resolution, specifically the use of longer delay times in the delayed-extraction ion source.