Carboxy-terminal sequencing: formation and hydrolysis of C-terminal peptidylthiohydantoins

Proteins and peptides can be sequenced from the carboxy terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory indicated that the use of trimethylsilyl isothiocyanate (TMS-ITC) as a coupling reagent significantly improved the yields and reaction conditions and reduced the number of complicating side products [Hawke et al. (1987) Anal. Biochem. 166, 298]. The present study further explores the conditions for formation of the peptidylthiohydantoins by TMS-ITC and examines the cleavage of these peptidylthiohydantoin derivatives into a shortened peptide and thiohydantoin amino acid derivative. Schizophrenia-related peptide (Thr-Val-Leu) was used as a model peptide and was treated with acetic anhydride and TMS-ITC at 50 degrees C for 30 min, and the peptidylthiohydantoin derivatives were isolated by reverse-phase HPLC and characterized by FAB-MS. The purified derivatives were subjected to a variety of cleavage conditions, and rate constants for hydrolysis were determined. Hydrolysis with acetohydroxamate as reported originally by Stark [(1968) Biochemistry 7, 1796] was found to give excellent cleavage of the terminal thiohydantoin amino acid, but also led to the formation of stable hydroxamate esters of the shortened peptide which are poorly suited for subsequent rounds of degradation. Hydrolysis with 2% aqueous triethylamine under mild conditions (1-5 min at 50 degrees C) was found to be more suitable for carboxy-terminal sequence analysis by the thiocyanate method. The shortened peptide, which could be isolated and subjected to a second round of degradation, and the released thiohydantoin amino acid are formed in good yield (90-100%). Several other small peptides containing 15 different C-terminal amino acid side chains were also investigated in order to examine any interfering reactions that might occur when these side chains are encountered in a stepwise degradation using the thiocyanate chemistry. Quantitative yields of peptidylthiohydantoins were obtained for all the amino acids examined with the following exceptions: low yields were obtained for C-terminal Glu or Thr, and no peptidylthiohydantoins were obtained for C-terminal Pro or Asp. Asparagine was found to form cyclic imides (64%) at the penultimate position (C-2) during hydrolysis of the peptidylthiohydantoins by 2% aqueous triethylamine. Cleavage of C-terminal Asn under these conditions led to the formation of the expected shortened peptide (69%), but also to the formation of a shortened peptide (31%) with a C-terminal amide. Problems with Glu and Thr could be solved by minimizing the reaction time with acetic anhydride.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of neutral amino-acid transport by isolated gill tissue of the bivalve Mya arenaria (L.)

An in vitro technique was used to examine the absorption by the gill of Mya arenaria (L.) of six neutral l-amino acids chosen for differences in their side chains, viz., short chain — glycine and alanine, long chain — leucine, sulphur containing — methionine, aromatic — phenylalanine, hydroxylic — serine. The uptake of all these substrates was active and carrier-mediated, and was analysed by Michaelis-Menten kinetics. Values of K_t, the transport constant, decreased with increasing length of the side chain for glycine, l-serine, l-alanine, l-methionine, l-phenylalanine, and l-leucine, while values for V_max, the maximum velocity of uptake, decreased as chain length increased, except in the case of l-serine. Inhibition experiments suggested that at least one transport locus was common to all the neutral amino acids examined, but homogeneity of transport was only demonstrated in the case of methionine and leucine. The transport of the basic amino acid l-lysine overlapped with several of the-neutral amino acids. These results emphasize the need to consider the mutual inhibitory effects between amino acids absorbed from sea water, when calculations are made of the value of this source of nutrition to marine invertebrates.     

The effects of hypothyroidism and fasting on electrogenic amino acid transfer: Possible evidence for multiple neutral amino acid carrier systems in rat jejunum

The jejunal mechanisms for the electrogenic transfer of four neutral amino acids (alanine, leucine, methionine, valine) and for sarcosine were characterised by an electrical method in vitro. The values for apparent K_m obtained electrically agree well with those assessed by conventional chemical techniques. Hypothyroidism and/or fasting rats for 3 days induced differential changes in the apparent K_m and p.d._max for the various amino acids. These alterations were interpreted as indicating the presence of at least three mechanisms for neutral amino acid transfer and one for sarcosine.In euthyroid rats, only alanine showed changes in apparent K_m (decrease) and p.d._max (decrease) after fasting for 3 days. With hypothyroidism the kinetic parameters of electrogenic transfer for alanine, valine and sarcosine were significantly altered while those for leucine and methionine were unaffected.     

A d-specific hydantoin amidohydrolase: properties of the metalloenzyme purified from Arthrobacter crystallopoietes

A d-specific hydantoinase has been purified to homogeneity from Arthrobacter crystallopoietes DSM 20117 with a yield of 5% related to the crude extract. The active enzyme is a tetramer of 257 kDa consisting of four identical subunits, each with a molecular mass of 60 kDa. Incubation of the enzyme with the metal-chelating agent EDTA had no inhibitory effect, while 8-hydroxyquinoline-5-sulfonic acid resulted in a complete and irreversible inactivation. The purified enzyme contains zinc as cofactor, which could be detected by subjection to direct analysis using inductive/coupled plasma-atomic emission spectrometry. The hydantoinase has a wide substrate specificity for the d-selective cleavage of 5-monosubstituted hydantoin derivatives with aliphatic and aromatic side chains. The V_max-value for phenylhydantoin is 217 U/mg, the K_m-value is 8 mM. Dihydrouracil was found to be a natural substrate (V_max=35 U/mg). The N-terminal amino acid sequence of the enzyme shows distinct homologies to other metal-dependent cyclic amidases involved in the nucleotide metabolism especially to dihydropyrimidinases as well as to ureases, l- and unselective hydantoinases. Due to these findings, this enzyme has to be considered as a possible link in the evolution to related l-selective and unselective hydantoinases from the genus of Arthrobacter.     

Changes in bulk protein of tobacco leaves on aerobic autolysis: Hydrogenation studies and identification of bound quinic acid

During aerobic autolysis and in commercial curing, the bulk proteins of tobacco leaves become coupled with quinic acid, presumably in consequence of coupling of chlorogenic acid congeners with lysine ε-NH₂ groups. Quinic acid derivatives, prepared from acid hydrolysates of such altered proteins, were identified by GC-MS. Such proteins were also hydrogenated over Rh/Al₂O₃ with a view to stabilizing the hypothetical linkages. Difficulties in removing contaminant Al had to be overcome. Evidence was then obtained (by GLC of derivatives) for several components, in acid hydrolysates of hydrogenated altered proteins, which were neither normal hydrogenation products of the common amino acids nor derivatives of quinic acid. Details of the chromatograms and mass spectra of quinic acid derivatives are provided in a supplementary publications.     

Analysis of adipimidate-crosslinked lysine

The chromatographic conditions for separation of N,N′-bislysyl(?-N)adipamidine and N-lysyl(?-N)adipamidinic acid, which were the products of acid hydrolysis of proteins treated with adipimidate esters, from other amino acids on an amino acid analyzer were established including their ninhydrin color values. Kinetics of decomposition of these lysine derivatives under the conditions of total acid hydrolysis of protein are also reported.     

Equilibrium dialysis binding studies of 1,N6-ethenoadenosine diphosphate (epsilon ADP) to myosin, heavy meromyosin, and subfragment one

The binding of the fluorescent analog of adenosine diphosphate (ADP)¹, 1,N⁶-ethenoadenosine diphosphate (εADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for εADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 (^± .5) × 10^?5 M^?1. This is identical to the values found by Young (J. Biol. Chem., $\text{242}$, 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of εATP versus ATP by S1 were studied. Values of V_max and K_m were 25 μM phosphate sec^?1 (gm protein)^?1 and 5 × 10^?5 M^?1 for ATP, and 80 μN phosphate sec^?1 (gm protein)^?1 and 45 × 10^?5 M^?1 for εATP. The results indicate that the increased V_max that occurs when εATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of εATP versus ADP. This in turn suggests that the increase in V_max may be due to an increased hydrolytic rate of εATP vs ATP in the enzyme substrate complex.     

Detection and characterization of a new beta-conglycinin from soybean seeds

A new protein has been isolated from the reserve proteins of the seeds of soybean (Glycine max) which is particularly deficient in methionine and cysteine. The protein dissociated in sodium dodecyl sulfate into a single polypeptide, M_r 48,000. The amino acid composition, N-terminal leucine and mobility on gel electrophoresis of this polypeptide all were indistinguishable from the β-subunit of β-conglycinin. In its nondissociated form, the protein behaved as a trimer of M_r, 137,000 ± 4000. Its sedimentation coefficient at ionic strength 0.5 was 7.5 S and it possessed antigenic determinants in common with β-conglycinin. This protein therefore has the properties of a new isomer of β-conglycinin—a homogeneous trimer of β subunits.     

Automated carboxy-terminal sequence analysis of peptides.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.     

Structural studies on epsilon-prototoxin of Clostridium perfringens type D. Localization of the site of tryptic scission necessary for activation to epsilon-toxin.

The mechanism of activation of ε-prototoxin to ε-toxin has been ascertained from partial amino acid sequences of both ε-prototoxin and ε-toxin. The activation of ε-prototoxin from $\text{Clostridium perfringens}$ type D by brief exposure to trypsin is caused by scission of a peptide bond between Lys₁₄ - Ala₁₅. A small peptide (14 amino acid residues) is split from the NH₂-terminus of the ε-prototoxin to give the active ε-toxin.     

Minimal nutrient requirements for induction of germination of Aspergillus niger conidia

Aspergillus niger reproduces asexually by forming conidia. Here, the minimal nutrient requirements were studied that activate germination of A. niger conidia. To this end, germination was monitored in time using an oCelloScope imager. Data was used as input in an asymmetric model to describe the process of swelling and germ tube formation. The maximum number of spores (P_max) that were activated to swell and to form germ tubes was 32.54% and 20.51%, respectively, in minimal medium with 50 mM glucose. In contrast, P_max of swelling and germ tube formation was <1% in water or 50 mM glucose. Combining 50 mM glucose with either NaNO₃, KH₂PO4, or MgSO4 increased P_max of swelling and germination up to 15.25% and 5.4%, respectively, while combining glucose with two of these inorganic components further increased these P_max values up to 25.85% and 10.99%. Next, 10 mM amino acid was combined with a phosphate buffer and MgSO₄. High (e.g. proline), intermediate and low (e.g. cysteine) inducing amino acids were distinguished. Together, a combination of an inducing carbon source with either inorganic phosphate, inorganic nitrogen or magnesium sulphate is the minimum requirement for A. niger conidia to germinate.     

Amino acid assimilation and respiration by attached and free-living populations of a marinePseudomonas sp.

The uptake kinetics of leucine and the assimilation and respiration of leucine, glycine, glutamate and arginine by a marinePseudomonas sp. was evaluated to determine whether the uptake and efficiency of substrate utilization of free-living bacteria differed from that of bacteria associated with surfaces. Bacteria were allowed to attach to plastic substrata with known hydrophilicities, as measured by advancing water contact angle (θ _A); these were Thermanox, poly(vinylidene fluoride), poly(ethelene) and poly(tetrafluoroethylene). The assimilation and respiration of surface-associated bacteria depended on the amino acid and substratumθ _A, but assimilation by surface-associated cells was generally greater than and respiration was generally less than that by free-living bacteria. The uptake kinetics with leucine demonstrated that the half saturation constant (K) of surface-associated bacteria was greater than that for free-living cells. The V_max values for surface-associated and free-living bacteria were similar, except for cells associated with poly(tetrafluoroethylene), which had a higher V_max value.     

Fasting and refeeding modulate neutral amino acid transport activity in the basolateral membrane of the rat exocrine pancreatic epithelium: fasting-induced insulin insensitivity

The effects of fasting and refeeding on amino acid transport in the perfused rat exocrine pancreas were investigated using a rapid dual tracer dilution technique. Unidirectional amino acid influx (15 s) was quantified (relative to the extracellular tracer d-mannitol) over a wide range of perfusate concentrations in pancreata isolated frm fed and 24 h, 48 h, and 72 h fasted and 72 h fasted and refed (24 h) animals. In fed animals transport of phenylalamine (1–24 mM) and l-serine (1–50 mM) was saturable and weighted non-linear regression analyses of the overall transport indicated an apparent K_t=10±3mM and V_max=7.0±1.0 μmol/min per g (n = 7) for phenylalanine and K_t=16±3 mM and V_max=20.6±2.1 μmol/min per g (n = 5) for serine. Fasting animals for 24 h or 48 h did not change the kinetics of either phenylalanine or serine transport. After a 72 h fast the rate of phenylalanine transport (V_max=15.9±2.9 μmol/min per g, (n = 5) was enhanced whereas the transport affinity (K_t=11±3 mM) remained unaltered. l-Serine transport was essentially unaltered. When 72 h fasted animals were refed for 24 h the V_max for the phenylalanine transport was reduced to values observed in fed animals. In parallel experiments refeeding had no significant effect on serine transport. Perfusion of pancreata isolated from 72 h fasted animals with bovine insulin (1 mU/ml or 1 μU/ml) did not stimulate either phenylalanine or serine transport. The fasting-induced stimulation of transport may provide a mechanism by which the extracellular supply of essential amino acids as phenylalanine is increased to meet the demands of continued proteolytic and lipolytic enzyme synthesis.     

Reactions of the amino groups of RNAse A: III. The existence of pH-dependent values of pK

The rates of the trinitrophenylation of the amino groups of ribonuclease A (RNAse) with the specific reagent trinitrobenzene sulfonic acid have been studied at 27°C, between pH 7.0 and 9.9. From the variation of the velocity constants with pH it has been shown that the reaction is biphasic in the sense that for each amino group two pKs have been found: one (pK = 7.3–7.52) in the range of pH between 7.0 and 8.3 and the other (pK = 9.28–9.69) in the pH range 8.5–9.9. It is pointed out that when the experimental conditions approached one another, there was agreement between the pK values obtained from titrimetric and kinetic studies. Evidence is presented from the literature concerning the validity of the pK value near 7.5 for the ε-amino groups in RNAse. The studies were repeated with performic acid oxidized RNAse and the 10 ε-amino groups were found to be monophasic with pK values between 8.01 and 8.10. The α-amino group of the N-terminal lysine was biphasic with a pK of 7.26 (pH range 7–8) and 8.13 (pH range 8.2–9.5).     

Sensitive,selective gas chromatographic—mass spectrometric analysis with trifluoroacetyl derivatives and a stable isotope for studying tissue sorbitol-producing activity

One of the major mechanisms involved in diabetic microangiopathy is considered to be an altered polyol pathway. However, clarifying the pathophysiology is difficult due to the lack of a sensitive method for measuring the reduction of glucose to sorbitol in tissue. Here we report a sensitive and selective method for polyol measurement using trifluoroacetyl (TFA) derivatives of polyols and stable isotope-labeled D-sorbitol (U-[¹³C]sorbitol, ¹³C₆H₁₄O₆, 98.7%) as an internal standard. Gas chromatography—mass spectrometry (GC—MS) using an SE-30 capillary column gave elution of TFA derivatives of sugars, polyols and U-[¹³C]sorbitol within 8 min, with clear separation of sorbitol. In the calibration study, the coefficients of correlation between the amount of sorbitol added and that determined in standard solutions containing 0.1–8.0 nmol sorbitol, erythrocyte mixture and liver cytosol mixture were r=0.999, r=0.997 and r=0.997, respectively. The precision of the GC—MS measurement of standard solution was C.V.=4.3%. Because glucose is used as a substrate, the method can clarify the polyol pathway under physiological conditions. With this method, K_m and V_max values of the reductase in erythrocytes were 115±19 mmol/l and 4.42±0.26 nmol/min/g of hemoglobin. In human liver, on the other hand, they were 75±132 mmol/l and 0.77±0.090 nmol/min/mg of protein, respectively. This difference of K_m values suggested that aldehyde reductase rather than aldose reductase is mainly responsible for reducing glucose to sorbitol in the liver. In conclusion, this newly developed method offers a highly sensitive and selective procedure for measuring low concentrations of sorbitol in various tissues and cells and should enable clarification of the kinetics of glucose reduction to sorbitol, which in turn can be used to evaluate the role of an altered polyol pathway in the pathophysiology of diabetic microangiopathy.     

Some Properties of Endo-polygalacturonase from Trichosporon penicillatum SNO-3

Some properties of the endo-polygalacturonase from Trichosporon penicillatum were investigated. The enzyme showed the highest activity around pH 5.0 and was stable at this pH up to 50°C. The enzyme catalyzed the hydrolysis of galacturonic acid oligomers as well as its polymer. The pentamer was degraded to a trimer and a dimer, the tetramer to a trimer and a monomer, and the trimer to a dimer and a monomer, respectively, whereas the dimer was not degraded. The kinetic constant V_max and Km values changed with the substrate chain-length; the Km values tended to decrease, whereas the V_max values tended to increase with increasing chain-length of the substrate. The amino acid residue participating in the active site of the enzyme was studied and it was found to be histidine.     

Isolation and Characterization of a Xylanase from Bacillus subtilis

Partial characterization of an extracellular xylanase isolated by chromatography from Bacillus subtilis gave a molecular weight of 32,000 and optimum pH and temperature of 5.0 and 50°C, respectively. K_m and V_max values, determined with a soluble larchwood xylan, were 0.16% and 7.0 × 10³ μmol min⁻¹ mg⁻¹ of enzyme respectively. The amino acid composition showed more basic amino acid residues than in a previously characterized xylanase from a white-rot fungus.     

The stimulatory effect of human chorionic gonadotropin on amino acid uptake by amphibian follicles.

Human chorionic gonadotropin (hCG) stimulates the uptake of eight different amino acids and four nucleosides by Xenopus laevis ovarian follicles. This hormone also stimulates amino acid uptake in the follicles of another amphibian, Callyptocephallela caudiverbera. The stimulation of uptake is due to a reduction in the amino acid concentration required for half-maximal uptake velocity and not to an increment in V_max. The effect of hCG does not require protein synthesis but requires physiological conditions of temperature and pH. Incorporation of radioactive exogenous amino acid into proteins is also stimulated by the hormone, but high-resolution electrophoresis shows that there are no drastic qualitative changes in the pattern of proteins synthesized at early times after hCG treatment. The effect of hCG on the uptake of exogenous amino acids does not appear to be required for oocyte maturation because other hormones such as progesterone and testosterone which induce maturation do not increase amino acid uptake. Also the concentration of hCG required for oocyte maturation is significantly lower than that required for an effect on amino acid transport. Inhibitors of oocyte maturation such as theophylline and cycloheximide do not inhibit the action of hCG on amino acid uptake by the amphibian follicles.     

Amino acid sequencing by gas chromatography-mass spectrometry using perfluoro-dideuteroalkylated peptide derivatives: A. Gas chromatographic retention indices

The gas chromatographic properties of more than 200 O-trimethylsilylated perfluoro-dideuteroalkyl polyamino alcohols were evaluated which were obtained by LiAlD₄-reduction and O-trimethylsilylation of N-perfluoroalkyl oligopeptide methyl esters. Complex derivatized mixtures were analyzed by gas chromatography-mass spectrometry which contained between 2 nmol and 12 μmol of the original peptides. Retention indices of these derivatives can be predicted by summation of retention index increments which are assigned to each amino acid residue. Upon application of a uniform factor, the predicted and experimentally found retention index values generally agree within ±30 units up to retention index 3000. Larger compounds, especially the heptafluoro derivatives, emerge significantly earlier than expected, which makes these the most useful derivatives of complex peptides.     

Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid Caffeoyltransferase From Tomato (Lycopersicon esculentum)

A novel acyltransferase from cotyledons of tomato (Lycopersicon esculentum Mill.), which catalyzes the transfer of caffeic acid from chlorogenic acid (5-O-caffeoylquinic acid) to glucaric and galactaric acids, was purified with a 2400-fold enrichment and a 4% recovery. The enzyme showed specific activities (theoretical V_max per milligram of protein) of 625 nanokatals (caffeoylglucaric acid formation) and 310 nanokatals (caffeoylgalactaric acid formation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it gave an apparent M_r of 40,000, identical to the value obtained by gel filtration column chromatography. Highest activity was found at pH 5.7, which was constant over a range of 20 to 120 millimolar K-phosphate. The isoelectric point of the enzyme was at pH 5.75. The reaction temperature optimum was at 38°C and the apparent energy of activation was calculated to be 57 kilojoules per mole. The apparent K_m values were 0.4 millimolar for glucaric acid, 1.7 millimolar for galactaric acid, and with both acceptors as second substrates 20 millimolar for chlorogenic acid. The relative ratio of the V_max/K_m values for glucaric acid and galactaric acid was found to be 100:12. Substrate-competition experiments support the conclusion that one single enzyme is responsible for both the glucaric and galactaric acid ester formation with marked preference for glucaric acid. It is proposed that the enzyme be called chlorogenic acid:glucaric acid O-caffeoyltransferase (EC 2.3.1.-). The three caffeic acid-dependent enzyme activities involved in the formation of the glucaric and galactaric acid esters, the chlorogenic acid:glucaric acid caffeoyltransferase as the key activity as well as the caffeic acid:CoA ligase and the caffeoyl-CoA:quinic acid caffeoyltransferase as the preceding activities, were determined. The time course of changes in these activities were followed during development of the seedling in the cotyledons and growth of the young plant in the first and second leaf. The results from tomato seedlings suggest a sequential appearance of these enzymes.