In vivo inhibition of aldehyde dehydrogenase by disulfiram

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not yet been determined. In this report, we demonstrate a novel, but simple and rapid method for structurally characterizing in vivo derived protein–drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (rmALDH) were purified in one-step with an affinity cartridge. The in vivo DSF-treated rmALDH showed 77% inhibition in enzyme activity as compared to that of the control. Subsequently, the control and DSF-inhibited rmALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited rmALDH as indicated by the mass increases of ∼71 and ∼100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH⁺=973.7 and 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC₃₀₁C₃₀₂C₃₀₃, derived from the enzyme active site region, and were modified at Cys₃₀₂ by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO).     

Overview--in vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC(50)=36.4 microM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC(50)=4.62 microM). We isolate and identify a carbamoylated peptide at Cys(302) with the sequence FNQGQC(301)C(302)C(303). Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys(302), present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.     

Overview — In vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC₅₀=36.4 μM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC₅₀=4.62 μM). We isolate and identify a carbamoylated peptide at Cys₃₀₂ with the sequence FNQGQC₃₀₁C₃₀₂C₃₀₃. Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys₃₀₂, present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.     

Inactivation of CMY-2 beta-lactamase by tazobactam: initial mass spectroscopic characterization.

The CMY-2 beta-lactamase, a plasmid determined class C cephalosporinase, was shown to be susceptible to inhibition by tazobactam (K(i)=40 microM). The reaction product(s) of CMY-2 beta-lactamase with the beta-lactamase inhibitor tazobactam were analyzed by electrospray ionization/mass spectrometry (ESI/MS) to characterize the prominent intermediates of the inactivation pathway. The ESI/MS determined mass of CMY-2 beta-lactamase was 39851+/-3 Da. After inactivating CMY-2 beta-lactamase with excess tazobactam, a single species, M(r)=39931+/-3.0, was detected. Comparison of the peptide maps from tryptic digestion of the native enzyme and the inactivated beta-lactamase followed by LC/MS identified two 22 amino acid peptides containing the active site Ser64 modified by a fragment of tazobactam. These two peptides were increased in mass by 70 and 88 Da, respectively. UV difference spectra following inactivation revealed the presence of a new species with a 302 nm lambda(max). Based upon the increase in molecular mass of the tazobactam inactivated CMY-2 beta-lactamase, we propose that during the inactivation of this beta-lactamase by tazobactam an imine is formed. Tautomerization forms the spectrally observed enamine. Hydrolysis generates the covalently attached malonyl semialdehyde, its hydrate, or an enol. This work provides information on the mass of a stable enzyme intermediate of a class C beta-lactamase inactivated by tazobactam and, for the first time, unequivocal evidence that a cross-linked species is not required for apparent inactivation.     

Effects of Angiotensin I-Converting Enzyme Inhibitory Substances Derived from Indonesian Dried-salted fish on Blood Pressure of Rats

Indonesian dried-salted fish (DSF) was produced from skipjack tuna by soaking the flesh in 15% NaCl (DSF I) or 25% NaCl (DSF II). The DSFs were then hydrolyzed by trypsin, chymotrypsin, Pronase E, and pepsin. Angiotensin I-converting enzyme (ACE) inhibitory activity was measured. The pepsin digest showed the highest inhibitory activity (IC₅₀; 0.63 mg protein/ml). DSF II hydrolysate had higher inhibitory activity than that in DSF I. A three-month storage period of DSF gave higher ACE-inhibitory activity than that of 6 months. An oral administration of pepsin hydrolysate significantly decreased the blood pressure of rats. From the purification steps, at least 4 inhibitor peptides were found. The amino acid sequences of the peptides were Val-Ala-Trp-Lys-Leu, Trp-Ser-Lys-Val-Val-Leu, Ser-Lys-Val-Pro-Pro, and Cys-Trp-Leu-Pro-Val-Tyr, with an IC₅₀ value of 31.97, 156.28, 74.22, and 22.20 μM, respectively.     

Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition

The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.     

ALDH1A1诱导肺腺癌细胞发生上皮-间质转化及化疗耐药

目的:探索醛脱氢酶1A1(aldehyde dehydrogenase 1A1,ALDH1A1)在肺腺癌细胞(lung adenocarcinoma cell,LAC)化疗耐药中的作用及机制,为肺癌临床治疗和新型药物的研发提供实验依据。方法:采用慢病毒载体构建ALDH1A1高表达肺腺癌细胞模型,并通过流式细胞术和western blot技术对该细胞模型进行验证。通过CCK8法检测ALDH1A1高表达肺腺癌细胞对肺癌治疗药物顺铂(cisplatin,DDP)、紫杉醇(paclitaxcel)、厄洛替尼(erlotinib)和吉非替尼(gefitinib)的耐药性。通过检测肿瘤干细胞(cancer stem cell,CSC)分子标志物、上皮-间质转化(Epithelial-Mesenchymal Transition,EMT)分子标志物及细胞迁移能力探讨ALDH1A1高表达对肺腺癌细胞的干性和EMT特征的影响。双硫仑(disulfiram,DSF)是ALDH的抑制剂,我们通过CCK8法和transwell细胞迁移实验探究DSF对肺腺癌细胞体外生长和迁移能力的影响,体内实验探究DSF和厄洛替尼联合用药对HCC827-ALDH1A1细胞皮下异种移植瘤生长的影响。结果:ALDH1A1高表达诱导肺腺癌细胞对厄洛替尼、吉非替尼、紫杉醇和顺铂产生不同程度的耐药,干细胞标志物CD44、CD133蛋白表达上调,EMT间充质标志物vimentin蛋白表达上调,transwell实验结果显示ALDH1A1高表达肺腺癌细胞的迁移能力增强,使用ALDH靶向抑制剂DSF能选择性抑制ALDH1A1高表达肺腺癌细胞所增高的迁移能力并克服HCC827-ALDH1A1细胞皮下异种移植瘤的生长,延缓体内耐药。结论:ALDH1A1能诱导肺腺癌细胞对多种抗肺癌药物产生耐药并发生干细胞样转化,靶向抑制ALDH酶活性可克服由ALDH1A1高表达所产生的耐药,为肺癌的临床治疗提供新的思路。     

Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry

A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.     

Identification of four novel types of in vitro protein modifications

In vitro chemical modifications in proteins, introduced during sample preparation, can complicate mass spectra and increase the potential for false-positive identifications. While several in vitro protein modifications have been described previously, additional types of such modifications may exist. Here, we report discovery of four types of in vitro protein modifications, identified by HPLC/MS/MS analysis and nonrestrictive protein sequence alignment by PTMap, an algorithm recently developed in our laboratory. These novel in vitro modifications included ethylation of aspartate and glutamate (+28 Da), esterification of aspartate and glutamate by glycerol (+74 Da), loss of 19 Da from lysine, and addition of 108 Da to cysteine. We confirmed that these modifications occurred in vitro and not in vivo in control experiments designed to avoid conditions likely to induce the modifications. We propose a plausible molecular mechanism for the -19 Da modification of lysine. Our study therefore conclusively identifies several novel in vitro protein modifications, suggests ways to avoid these modifications, and highlights the possibility of misidentification of peptides because of in vitro modifications.     

P450 active site architecture and reversibility: inactivation of cytochromes P450 2B4 and 2B4 T302A by tert-butyl acetylenes

The inactivations of P450 2B4 and the T302A mutant of 2B4 by tert-butyl acetylene (tBA) and the inactivation of 2B4 T302A by tert-butyl 1-methyl-2-propynyl ether (tBMP) have been investigated. tBA and tBMP inactivated both enzymes in a mechanism-based manner with the losses in enzymatic activity corresponding closely to losses in P450 heme. HPLC and ESI-LC-MS analysis detected two different tBA- or tBMP-modified heme products with masses of 661 and 705 Da, respectively. Interestingly, the inactivations of the P450s 2B4 by tBA and tBMP were partially reversible by dialysis, and the tBA- or tBMP-modified heme products could only be observed with ESI-LC-MS/MS when the inactivated samples were acidified prior to analysis, indicating a requirement for protons in the formation of stable heme adducts in both the wild-type and mutant 2B4 enzymes. Results of studies using artificial oxidants to support enzyme inactivation suggest that the oxenoid-iron activated oxygen species is preferentially utilized during the inactivation of the P450s 2B4 by tBA. These results argue against the use of a peroxo-iron species by P450 2B4 T302A. Molecular dynamics studies of wild-type P450 2B4 reveal that contiguous hydrogen bond networks, including structural waters, link a conserved glutamate (E301) to the distal oxygen of the peroxo-heme species via threonine 302. Interestingly, models of 2B4 T302A reveal that a compensatory, ordered hydrogen bond network forms despite the removal of T302. These results indicate that while T302 may play a role in proton delivery in the formation of the oxenoid-iron complex and in the stabilization of acetylene heme adducts in 2B4, it is not essential for proton delivery given the presence of E301 in the binding site.     

Thymidine metabolism in cells treated with DNA-suppressing factor (DSF)

Inhibition of thymidine incorporation into DNA in cells treated with DNA-suppressing factor (DSF) has been studied. After 16 hr treatment with DSF, transport of labeled thymidine across the cell membrane was not inhibited, since equilibrium of labeled thymidine with the acid-soluble pool occurred at the same rate and the radioactivity was at the same level as in untreated cells. The values of Vmax and Km in the kinetics of transport of exogenous thymidine were not changed by DSF. Phosphorylation of labeled thymidine to deoxythymidine triphosphate (dTTP) was not inhibited by DSF. After a chase of labeled thymidine, radioactivity of the acid-soluble fraction in DSF-treated cells decreased more rapidly but that of the acid-insoluble fraction remained at a lower level than in untreated cells. It was assumed that DSF might block the entry of dTTP into DNA.     

Discrimination of in vitro and in vivo digestion products of meat proteins from pork,beef, chicken,and fish

In vitro digestion products of proteins were compared among beef, pork, chicken, and fish. Gastric and jejunal contents from the rats fed these meat proteins were also compared. Cooked pork, beef, chicken, and fish were homogenized and incubated with pepsin alone or followed by trypsin. The digestion products with molecular weights of less than 3000 Da were identified with MALDI‐TOF‐MS and nano‐LC‐MS/MS. Gastric and jejunal contents obtained from the rats fed the four meat proteins for 7 days were also analyzed. After pepsin digestion, pork, and beef samples had a greater number of fragments in similarity than chicken and fish samples, but the in vitro digestibility was the greatest (p < 0.05) for pork and the smallest for beef samples. After trypsin digestion, the species differences were less pronounced (p > 0.05). A total of 822 and 659 peptides were identified from the in vitro and in vivo digestion products, respectively. Our results could interpret for the differences in physiological functions after the ingestion of different species of meat.     

Comprehensive identification of novel post-translational modifications in cellular peroxiredoxin 6

Peroxiredoxin 6 (PRDX6), a 1-Cys peroxiredoxin, is a bifunctional enzyme acting both as a glutathione peroxidase and a phospholipase A2. However, the underlying mechanisms and their regulation mechanisms are not well understood. Because post-translational modifications (PTMs) have been shown to play important roles in the function of many proteins, we undertook, in this study, to identify the PTMs in PRDX6 utilizing proteomic tools including nanoUPLC-ESI-q-TOF MS/MS employing selectively excluded mass screening analysis (SEMSA) in conjunction with MOD(i) and MODmap algorithm. We chose PRDX6 obtained from liver tissues from two inbred mouse strains, C57BL/6J and C3H/HeJ, which vary in their susceptibility to high-fat diet-induced obesity and atherosclerosis, and a B16F10 melanoma cell line for this study. When PRDX6 protein samples were separated on 2D-PAGE based on pI, several PRDX6 spots appeared. They were purified and the low abundant PTMs in each PRDX6 spot were analyzed. Unexpected mass shifts (Δm = -34, +25, +64, +87, +103, +134, +150, +284 Da) observed at active site cysteine residue (Cys47) were quantified using precursor ion intensities. Mass differences of -34, +25, and +64 Da are presumed to reflect the conversion of cysteine to dehydroalanine, cyano, and Cys-SO(2) -SH, respectively. We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da) as well as unknown modifications (+134, +150, +284 Da). Comprehensive analysis of these PTMs revealed that the PRDX6 exists as a heterogeneous mixture of molecules containing a multitude of PTMs. Several of these modifications occur at cysteine residue in the enzyme active site. Other modifications observed, in PRDX6 from mouse liver tissues included, among others, mono- and dioxidation at Trp and Met, acetylation at Lys, and deamidation at Asn and Gln. Comprehensive identification of the diverse PTMs occurring in this bifunctional PRDX6 enzyme should help understand how PRDX6 plays key roles in oxidative stresses.     

Production and Isolation of Levan by Use of Levansucrase Immobilized on the Ceramic Support SM-10

The complete amino acid sequence of rye seed chitinase-a (RSC-a) has been analyzed. RSC-a was cleaved with cyanogen bromide and the resulting three fragments, CB1, CB2, and CB3, were separated by gel filtration. The amino acids of the N-terminal fragment CB1 were sequenced by analyzing the peptides produced by digestion with trypsin, lysylendopeptidase, or pepsin of reduced S-carboxymethyl ated or S-aminoethylated CB1. The sequences of fragments CB2 and CB3 were established by sequencing the tryptic peptides from reduced S-carboxymethylated CB2 and CB3, and by aligning them with the sequence of rye seed chitinase-c (RSC-c) to maximize sequence homology. The complete amino acid sequence of RSC-a was established by connecting these three fragments.

RSC-a consists of 302 amino acid residues including hydroxyproline residues, and has a molecular mass of 31,722 Da. RSC-a is basic protein with a cysteine-rich amino terminal domain, indicating that this enzyme belongs to class I chitinases. The amino acid sequence of RSC-a showed that the sequence from Gly60 to C-terminal Ala302 in this enzyme corresponds to that of RSC-c belonging to class II chitinases with 92% identity, and that RSC-a has high similarity to other plant class I chitinases but a longer hinge region and an extra disulfide bond.     

A systematical analysis of tryptic peptide identification with reverse phase liquid chromatography and electrospray ion trap mass spectrometry

In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identified peptides in reverse phase liquid chromatography and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Following protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identified peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol.Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state ( 2, 3) peptides are likely to be identified, and the hydropathicity of the peptides might not be related to whether it is more likely to be identified or not. The number of identified peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identified peptides present three types of redundancy, namely identification, charge, and sequence redundancy, which may repress low abundance protein identification.     

Inhibition of hepatic aldehyde dehydrogenases in the rat by calcium carbimide (calcium cyanamide)

Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration.     

Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates

The in vitro DPP-IV inhibitory activity of isolated peptides from of tuna cooking juice hydrolyzed by Protease XXIII (PR) and orientase (OR) was determined. The results showed that the peptide fractions with the molecular weight over 1,422 Da possessed the greatest DPP-IV inhibitory activity. The amino acid sequences of the three peptides isolated from PR and OR hydrolysates were identified by MALDI-TOF/TOF MS/MS, and they were Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu (1412.7 Da), Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg (1690.8 Da) and Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.6 Da), while they showed the dose-dependent inhibition effect of DPP-IV with IC(50) values of 116.1, 78.0 and 96.4 μM, respectively. In vitro simulated gastrointestinal digestion retained or even improved the DPP-IV inhibitory activities of the three peptides. The results suggest that tuna cooking juice would be a good precursor of DPP-IV inhibitor, and the DPP-IV inhibitory peptides can successfully passed through the digestive tract.     

Isotopically labeled crosslinking reagents: resolution of mass degeneracy in the identification of crosslinked peptides

Mass spectrometry in three dimensions (MS3D) is a newly developed method for the determination of protein structures involving intramolecular chemical crosslinking of proteins, proteolytic digestion of the resulting adducts, identification of crosslinks by mass spectrometry (MS), peak assignment using theoretical mass lists, and computational reduction of crosslinks to a structure by distance geometry methods. To facilitate the unambiguous identification of crosslinked peptides from proteolytic digestion mixtures of crosslinked proteins by MS, we introduced double 18O isotopic labels into the crosslinking reagent to provide the crosslinked peptides with a characteristic isotope pattern. The presence of doublets separated by 4 Da in the mass spectra of these materials allowed ready discrimination between crosslinked and modified peptides, and uncrosslinked peptides using automated intelligent data acquisition (IDA) of MS/MS data. This should allow ready automation of the method for application to whole expressible proteomes.