Negative ion fragmentation of cysteic acid containing peptides: cysteic acid as a fixed negative charge.

We present here a study of the collision induced dissociation (CID) of deprotonated cysteic acid containing peptides produced by MALDI. The effect of cysteic acid (C(ox)) position is interrogated by considering the positional isomers, C(ox)LVINVLSQG, LVINVLSQGC(ox), and LVINVC(ox)LSQG. Although considerable variation between the CID spectra is observed, the mechanistic picture that emerges involves charge retention at the deprotonated cysteic acid side chain. Fragmentation occurs in the proximity of the cysteic acid group by charge directed mechanisms as well as remote from this group to form ions, which may be rationalized by charge remote mechanisms. Additionally, the formation of the SO(3)(-•) ion is observed in all cases. Fragmentation of C(ox)LVINVLSQC(ox) provides both N- and C-terminal, y and b ions, respectively indicating that the negative charge may be retained at either of the cysteic acids; however, there is some evidence that charge retention at the C-terminal cysteic acid may be preferred. Fragmentation of tryptic type peptides containing a C-terminal arginine or lysine residue is considered through comparison of three peptides C(ox)LVINKLSQG, C(ox)LVINVLSQK, and C(ox)LVINVLSQR. Lastly, we rationalize the formation of b(n-1)+ H(2)O and a(n-1) ions through a mechanism involving rearrangement of the C-terminal residue to form a mixed anhydride intermediate.

N-Fmoc-α-sulfo-ß-alanine: a versatile building block for the water solubilisation of chromophores and fluorophores by solid-phase strategy.

An easy and efficient solid-phase synthesis strategy to obtain rapidly water-soluble chromophores/fluorophores in highly pure form has been developed. This first successful use of N-Fmoc-α-sulfo-ß-alanine as a SPPS building block opens the way to the future development of promising direct "on-resin" peptide labelling and water-solubilising methods.

Mucin-producing carcinoma of the gallbladder associated with primary sclerosing cholangitis and ulcerative colitis.

Mucin-producing carcinoma of the gallbladder is very rare. We report here a case of mucin-producing carcinoma of the gallbladder associated with primary sclerosing cholangitis (PSC) and ulcerative colitis (UC). A 74-year-old female had been treated with salazosulfapyridine and ursodesoxycholic acid becase of UC and PSC. After 7 years of treatment, laboratory data showed that the liver function took a turn for the worse, and the patient was admitted to our hospital for further examination. Enhanced computed tomography and ultrasonography showed an enlarged gallbladder associated with wall thickening and diffuse papillary protrusion. Endoscopic retrograde cholangiography showed stenosis and dilatation of the bile duct, which were compatible with PSC. Under the diagnosis of an early carcinoma of the gallbladder, we performed simple cholecystectomy. The tumor showed a papillary growth pattern located diffusely in the gallbladder with a massive amount of mucin filling the gallbladder. Histologically, it was diagnosed as a papillary adenocarcinoma localized in the mucosal layer. To the best of our knowledge, this is the first case of mucin-producing carcinoma of the gallbladder associated with PSC and UC. PSC and UC patients should be regarded as a high-risk group not only for cholangiocarcinoma but also carcinoma of the gallbladder.

Effects of choleretics on bile compositions drained from patients with pigment gallstone.

OBJECTIVE: To provide evidence for three-level prevention of cholelithiasis by means of observing the effects of some choleretics on bile compositions drained from patients with pigment gallstone. METHODS: Twenty-seven patients suffering from primary pigment gallstones and having received treatment of choledochostomies plus T-tube or endoscopic nasal bile drainage (ENBD) were divided equally into three groups, and administered respectively with Lidanling (the LDL group), ursodesoxycholic acid (the UDA group) and combination of LDL and UDA (the LDL + UDA group) through oral intake (7 patients in each group). Besides, 6 post-operational patients got no treatment with any drug were allocated in the control group. Bile of all the patients was collected before treatment and on the 1, 3, 5, 7 th day after the treatment started to detect levels of total bile acid (TBA), glycocholic acid (GCA), taurocholic acid (TCA), glycocholic cheno-desoxycholic acid (GCDCA), total bilirubin (TBIL), uncombined bilirubin (UCB), concentration of calcium ion (Ca(2+)) as well as the bacterio-genetic and endogenous beta-glucuronidase activity for comparing. RESULTS: Levels of TBA, GCA, TCA and GCDCA got gradually increased in the UDA group and the LDL + UDA group after treatment (P < 0.05), while those in the LDL group remained unchanged, showing an insignificant difference as compared with those in the control group. In the LDL group and the LDL + UDA group, TBIL gradually increased while UCB gradually decreased in the course of treatment (P < 0.05). Moreover, levels of Ca(2+) and endogenous beta-glucuronidase activity got significantly lowered (P < 0.05). CONCLUSION: Combined use of LDL and UDA could elevate levels of TBA, GCA, TCA, GCDCA, enhance the excretion of TBIL in patients with pigment gallstone after bile drainage, lower levels of UCB and Ca(2+) and the activity of endogenous beta-glucuronidase in the bile, so as to reduce the possibility of stone formation of bile, and therefore, it could be used to prevent the production of pigment gallstone, especially to prevent post-operative recurrence of stones.

High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry.

A high-throughput method for sequencing of N termini of proteins by using postsource decay (PSD) of matrix-assisted laser desorption/ionization mass spectrometry has been developed. After a protein blotted on the PVDF membrane was successively reduced, S-alkylated, and guanidinated, its N-amino group was coupled to biotinylcysteic acid. The protein was then extracted from the membrane and digested with trypsin. The derivatized N-terminal fragment was then specifically isolated from the tryptic digest with avidin resins, and its de novo sequencing was successfully performed by PSD utilizing a sulfonic acid group introduced to the N terminus.

Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing.

Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following beta-elimination/sulfite addition to convert -HPO4 to -SO3 has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na2SO3/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 microM concentration of the peptide. Both cysteic acid and beta-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.

Electrophoretic studies on the chelating tendency of bioactive sulphur-containing amino acids. The metal-methylcysteine-cysteine system.

Stability constants of binary Fe(III)-methylcysteine, Cr(III)-methylcysteine and mixed Fe(III)-methylcysteine-cysteine, Cr(III)-methylcysteine-cysteine complexes have been determined by paper electrophoresis at 0.1 M ionic strength and a temperature of 35 degrees C. The stability constants of Fe(III)-methylcysteine-cysteine and Cr(III)-methylcysteine-cysteine mixed complexes were found to be 6.00 +/- 0.07 and 5.05 +/- 0.15 (logarithm of stability constant values), respectively.

Effects of newly synthesized analogs of MIF-1 containing unnatural amino acids on electrically evoked smooth muscle contractions.

New MIF-1 (Pro-Leu-Gly-NH2) analogs containing unnatural amino acids such as L-canavanine (Cav) and L-cysteic acid S-(2-aminoethyl)amide (sLys) have been synthesized and in vitro experiments were performed to study their action on neurotransmission in target tissues with adrenergic and cholinergic neurotransmission. The experiments were carried out on electrically stimulated proximal guinea pig ileum (GPI) and the prostatic part of rat and rabbit vasa deferentia (VDR, VDRabb). The present results show that the newly synthesized Cav2-MIF and sLys2-MIF might affect electrically evoked smooth muscle contractions.

Penicillin biosynthesis: intermediates of biosynthesis of delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine formed by ACV synthetase from Acremonium chrysogenum.

The tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) is synthesised by the multifunctional enzyme ACV synthetase integrating four steps of the penicillin and cephalosporin biosynthetic pathway. Peptide synthesis follows the thiotemplate mechanism from intermediates bound as thioesters to the enzyme. The formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-thioester in the absence of L-valine was shown by isolation of the enzyme-substrate complex and cleavage of the covalently bound intermediate with performic acid. The dipeptide was recovered as cysteic acid or cysteic acid oxime and detected by HPLC and MALDI-TOF mass spectrometry. We conclude that the first peptide bond is formed between delta-carboxyl of L-aminoadipic acid and L-cysteine, followed by addition of the dipeptidyl intermediate to L-valine.

In vitro study of the antiseborrheic activity of zinc L-cysteate, a novel zinc compound, on rat preputial gland.

The antiseborrheic effect of zinc L-cysteate, a new zinc compound, was evaluated in vitro by determining the lipidic metabolic activity of rat preputial glands as measured by incorporation of 14C-sodium acetate. At 10(-3) and 10(-4) mol/l, zinc L-cysteate was more active than S-carboxymethyl L-cysteine used as reference in corresponding concentrations (p < 0.05 and p < 0.01, respectively). The pharmacologic results seem promising for clinical studies in dermatology.

Hypocholesterolemic effect of ursodeoxycholylcysteic acid in hamsters fed a high cholesterol diet.

We studied the effect of feeding ursodeoxycholylcysteic acid, the cysteic acid conjugated analog of ursodeoxycholyltaurine, on serum and liver cholesterol levels and on intestinal absorption of cholesterol and bile salts in hamsters. Addition of ursodeoxycholylcysteic acid to the cholesterol-enriched diet reduced the elevation of serum and liver cholesterol levels caused by feeding cholesterol. However, supplementation with ursodeoxycholylcysteic acid to the standard diet did not show any significant change in serum and liver cholesterol levels. Administration of ursodeoxycholylcysteic acid caused a decrease in dietary cholesterol absorption but did not interfere with the ileal transport of endogenous bile salts. Hence the hypocholesterolemic activity of dietary ursodeoxycholylcysteic acid is thought to be the effect on intestinal absorption of cholesterol but not to be the interruption of the enterohepatic circulation of bile salts.

Reversed-phase high-performance liquid chromatographic separation and quantitation of phenylthiohydantoin derivatives of 25 amino acids, including those of cysteic acid, 4-hydroxyproline, methionine sulfone, S-carboxymethylcysteine and S-methylcysteine.

A high-performance liquid chromatography system is presented which allows separation and quantitation (in the range 4-1000 pmol) of all common phenylthiohydantoin amino acids, including derivatives of 4-hydroxyproline, methionine sulfone and three differently modified forms of cysteine. By showing the actual solvent gradient during elution (as opposed to the programmed gradient) and by supplying information on the effects of minor changes in solvent-pH, column temperature, flow-rate, and concentration of 2-propanol in the gradient, we make guidelines available for fine-tuning the separation with new Ultrasphere-ODS (C18) columns.

[Thermodynamic and kinetic aspects of the hydrolysis of ethyl(R)-2- (benzyloxycarbonylamino)-3-sulfamoylpropionate in the presence of free and immobilized alpha-chymotrypsin]. / Thermodynamische und kinetische Aspekte der Hydrolyse des (R)-2- (Benzyloxycarbonylamino)-3-sulfamoylpropionsäure-ethylesters in Gegenwart von freiem und trägergebundenem alpha-Chymotrypsin.

The hydrolysis of ethyl (R)-2-(benzyloxycarbonylamino)-3-sulfamoylpropionate (blocked cysteic acid S-amide) by native and immobilized alpha-chymotrypsin was studied. The experiments were performed using a constant enzyme/substrate ratio of 1:8 and at a temperature of 10-40 degrees C; the immobilized enzyme was bound to a dialdehyde cellulose matrix. A kinetic equation (Eq.10) was found to be applicable which confirms that the mechanism of the enzyme reaction consists of several stages, irrespective of the enzyme state. The temperature dependence of the reaction velocity was investigated and applied using the Arrhenius equation. The constant value thus obtained for the activating energy showed that the active centres retained their character during immobilization. The differences between the velocities of the reaction with immobilized and with native enzyme corresponded to the different number of active centres during the reaction time. Based on these results a kinetic model of the mechanism of the studied reaction is presented which includes an initial balanced stage of the chemosorption type.

Studies on the action mechanism of the antihemostatic effect of lodopeptides.

A synthetic iodopeptide having a glutamic acid-diiodotyrosine molar ratio of 1:1 has been shown to be an effective anticoagulant both in vivo and in vitro. Contrasted with heparin the following general conclusions may be made regarding its action. The iodopeptide does not act through the inactivation of thrombin in plasma. Iodopeptide does interact with fibrinogen to form a complex which, in vitro, is not soluble in buffered saline at physiological pH. At pH 8, iodopeptide interacts with fibrinogen to form a soluble complex in the presence of 0.9% NaCl that is not coaguable either by thrombin or Crotalus venom enzymes. All the available evidence indicates that the fibrinogen to fibrin conversion is not inhibited under these conditions, but that fibrin, once formed, is not able to polymerize due to interference by iodopeptide. Similar results were obtained with heparin in vitro with thrombin-fibrinogen mixtures in the absence of NaCl. Studies with Russell's viper venom in native PRP strongly suggest that the iodopeptide also interferes with processes in the early coagulation pathway associated with prothrombin activation.