Quantitative polyphosphate-induced “prebiotic” peptide formation in H2O by addition of certain azoles and ions

Summary Aqueous solutions of 0.1 M amino acid and 0.1 M trimetaphosphate maintained at chosen pH values between 8.0 and 10.5 and at room temperature in the presence of imidazole or 1,2,4-triazole give rise after a few days to the corresponding peptides. Yields are highest when the pH is adjusted with concentrated NaOH or KOH instead of ammonia; in some cases glycine is quantitatively transformed within 10–15 days into peptides, mainly diand tripeptides.     

Proteases of nuclei of testicular cells assayed with fluorogenic peptides

Nuclei of seminiferous epithelial cells (SEC) of rat testis exhibit protease activity when assayed with fluorogenic peptides or with [3H]histones. At pH 8 the nuclear protease rapidly hydrolyzes BOC-Val-Pro-Arg-7-Amino-4-Methyl Coumarin (BVPAC) and Glu-Gly-Arg-7-Amino-4-Methyl Coumarin (GGAC), but this enzyme does not hydrolyze CBZ-Arg-7-Amino-4-Methyl Coumarin (CAC) or Glu-Phe-7-Amino-4-Methyl Coumarin (GPC). The cytoplasm of these cells hydrolyzes each of these substrates. The protease activity versus BVPAC can be extracted from cytoplasm and nuclei with 0.1 M H2SO4. The extracted activity from cytoplasm is lost during storage for 5 days at either pH 3 or pH 8 at -25 degrees, but the activity extracted from nuclei is maintained under these conditions. The nuclear protease activity is found in SEC of young rats prior to the appearance of acrosin.     

An improved protocol for coupling synthetic peptides to carrier proteins for antibody production using DMF to solubilize peptides.

We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.     

Bacillus thuringiensis parasporal crystal toxin: Dissociation into toxic low molecular weight peptides

Parasporal crystals of Bacillus thuringiensis can be dissociated into low molecular weight peptides (< 5000 daltons) by dissolving them in 0.1 M N-morpholinopropane sulfonic acid buffer pH 7.8 containing 0.05 M dithiothreitol and 2M–4M KSCN, or by performic acid oxidation. The peptides obtained by dissolving in KSCN were still toxic to silkworm larvae.     

Peptides are building blocks of heat-induced fibrillar protein aggregates of beta-lactoglobulin formed at pH 2

The proteinaceous material present in beta-lactoglobulin fibrils formed after heating (20 h at 85 degrees C) at pH 2 was identified during this study. Fibrils were separated from the nonaggregated material, and the fibrils were dissociated using 8 M guanidine chloride and 0.1 M 1,4-dithiothreitol (pH 8). Characterization of the different fractions was performed using thioflavin T fluorescence, high-performance size-exclusion chromatography, reversed-phase HPLC, and mass spectrometry (MALDI-TOF). Beta-lactoglobulin was found to be hydrolyzed into peptides with molecular masses between 2000 and 8000 Da, and the fibrils were composed of a part of these peptides and not intact beta-lactoglobulin. The majority of the peptides (both aggregated and nonaggregated) were a result from cleavage of the peptide bonds before or after aspartic acid residues. Explanations for the presence of certain peptide fragments in the fibrils are the hydrophobicity, low charge, charge distribution, and capacity to form beta-sheets.     

Valinomycin inhibited methane synthesis in Methanobacteriumthermoautotrophicum

$\text{Methanobacterium}\text{thermoautotrophicum}$ cells, incubated anaerobically under H₂ in 0.1 M KCl or 0.1 M NaCl, above pH 7.5, are interior acid with respect to the incubation medium. The pH gradient thus established can be discharged by either carbonyl cyanide m-chlorophenylhydrazone or valinomycin at high concentration (17μM). In these cells, which actively synthesize CH₄ from CO₂ and H₂, methanogenesis is strongly inhibited when the pH gradient is discharged.     

Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect

Data are reported for T(m), the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2-9) and salt concentration (range 0-1 M) for two salts, Na(2)SO(4) and NaCl. The results show stabilization by sulfate via anion-specific binding in the concentration range 0-0.1 M and via the Hofmeister effect in the concentration range 0.1-1.0 M. The increase in T(m) caused by anion binding at 0.1 M sulfate is 20 degrees at pH 2 but only 1 degree at pH 9, where the net proton charge on the protein is near 0. The 10 degrees increase in T(m) between 0.1 and 1.0 M Na(2)SO(4), caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH-dependent stabilization is produced by 1 M NaCl: the increase in T(m) between 0 and 1.0 M is 14 degrees at pH 2 but only 1 degree at pH 9. The 14 degree increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na(2)SO(4) and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride.     

Ethylene promotes the induction by auxin of the cortical microtubule randomization required for low-pH-induced root hair initiation in lettuce (Lactuca sativa L.) seedlings

Transverse cortical microtubule (CMT) arrays in lettuce root epidermal cells randomize soon after a shift from pH 6.0 to pH 4.0, and this randomization is essential for root hair initiation. We investigated the hormonal regulation of CMT randomization. At pH 4.0, 1 micro M of the auxin competitive inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid (PCIB), 0.1 micro M of the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) or 0.1 micro M of the ethylene action inhibitor Ag(+) suppressed CMT randomization and root hair initiation. At pH 6.0, addition of 0.1 micro M indole-3-acetic acid (IAA) or 1 micro M of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) induced CMT randomization and root hair initiation. Culturing with 0.1 micro M IAA plus 0.1 micro M AVG, or 1 micro M ACC plus 1 micro M PCIB also induced these phenomena. ACC (1 micro M) plus 100 micro M PCIB inhibited CMT randomization and root hair initiation, but 1 micro M AVG with 0.1 micro M Ag(+) and 0.1 micro M IAA induced them. These results suggest that auxin is essential for CMT randomization. As a higher concentration of PCIB was required to suppress CMT randomization when ACC was added, the greater amount of ethylene produced at pH 4.0 may promote the induction by auxin of CMT randomization in hair-forming cells.     

A model-dependent approach to correlate accelerated with real-time release from biodegradable microspheres

The purpose of this study was to determine the feasibility of applying accelerated in vitro release testing to correlate or predict long-term in vitro release of leuprolide poly(lactideco-glycolide) microspheres. Peptide release was studied using a dialysis technique at 37°C and at elevated temperatures (50°C–60°C) in 0.1 M phosphate buffered saline (PBS) pH 7.4 and 0.1 M acetate buffer pH 4.0. The data were analyzed using a modification, of the Weibull equation. Peptide release was temperature dependent and complete within 30 days at 37°C and 3 to 5 days at the elevated temperatures. In vitro release profiles at the elevated temperatures correlated well with release at 37°C. The shapes of the release profiles at all temperatures were similar. Using the modified Weibull equation, an increase in temperature was characterized by an increase in the model parameter, α, a scaling factor for the apparent rate constant. Complete release at 37°C was shortened from ∼30 days to 5 days at 50°C, 3.5 days at 55°C, 2.25 days at 60°C in PBS pH 7.4, and 3 days at 50°C in acetate buffer pH 4.0. Values for the model parameter β indicated that the shape of the release profiles at 55°C in PBS pH 7.4 (2.740) and 50°C in 0.1 M acetate buffer pH 4.0 (2.711) were similar to that at 37°C (2.577). The E_a for hydration and erosion were determined to be 42.3 and 19.4 kcal/mol, respectively. Polymer degradation was also temperature dependent and had an E_a of 31.6 kcal/mol. Short-term in vitro release studies offer the possibility of correlation with long-term release, thereby reducing the time and expense associated with longterm studies. Accelerated release methodology could be useful in the prediction of long-term release from extended release microsphere dosage forms and may serve as a quality control tool for the release of clinical or commercial batches. Selected for the 2005 AAPS Outstanding Graduate Student Research Award in Pharmaceutical Technologies Sponsored by Solvay Pharmaceuticals.     

Effect of vasoactive intestinal peptide, peptide histidine isoleucine and growth hormone-releasing factor-40 on bombesin-like immunoreactivity, somatostatin and gastrin release from the perfused rat stomach

Bombesin-like immunoreactivity (BLI) has been demonstrated in neurons of the gastrointestinal tract and gastric BLI secretion can be demonstrated in response to the classical neurotransmitter acetylcholine. Since structurally related peptides VIP, PHI and GRF have to be considered as peptidergic neurotransmitters it was of interest to determine their effect on gastric BLI secretion. Additionally, somatostatin (SLI) and gastrin secretion was examined. The isolated stomach of overnight fasted rats was perfused with Krebs-Ringer buffer via the celiac artery and the effluent was collected via the portal vein. The gastric lumen was perfused with isotonic saline at pH7 or pH2. All four peptides were tested at a dose of 10(-11) M and 10(-8) M at both pH levels and in addition the effect of VIP and PHI was examined at 10(-14) M and 10(-12) M during luminal pH2. At luminal pH7 VIP and PHI stimulated SLI release at 10(-8) M but had no effect on BLI or gastrin secretion. rGRF and hpGRF were both ineffective on SLI and gastrin release while rGRF inhibited and hpGRF stimulated BLI secretion. This effect was not dose related. At luminal pH2 all four peptides stimulated BLI secretion. Stimulation by PHI was already observed at a dose of 10(-14) M while VIP elicited a stimulatory effect at 10(-12) M. PHI at the two lowest concentrations of 10(-14) and 10(-12) M elicited a stimulation of SLI and gastrin release while the same doses of VIP and the higher doses of all four peptides had no effect on SLI and gastrin secretion at an acidic intraluminal pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of fixation for immunohistochemistry on tissue concentrations of substance P and somatostatin determined by radioimmunoassay

Concentrations of substance P and somatostatin were measured in preparations of the myenteric plexus (plus longitudinal muscle) of the guinea-pig ileum after fixation and processing for immunohistochemistry and compared with concentrations measured in fresh tissue. Two fixative solutions were used: (i) 4% formalin in phosphate buffer (0.1 M, pH 7.0); and (ii) a mixture of aqueous picric acid with 2% formalin in phosphate buffer (0.1 M, pH 7.0). Tissues were extracted in boiling aqueous acetic acid (2.0 M) either immediately after fixation and processing or after storage for up to four weeks in phosphate-buffered saline (PBS) with or without sodium azide. The concentrations of substance P and somatostatin in these extracts were measured by radioimmunoassay and compared to the concentrations in extracts of fresh tissue. The concentration of substance P in fixed tissue was the same as that found in fresh tissue, whereas the concentration of somatostatin in fixed tissue was half that found in fresh tissue (P<0.01). If the tissue was not subjected to the extensive washing for immunohistochemistry, somatostatin concentrations in fresh and fixed tissue were not significantly different. The concentration of substance P did not change on storage of the fixed tissue in PBS, either with or without sodium azide. The concentration of somatostatin decreased on storage of the fixed tissue in PBS over four weeks to 40% of its original value, but the presence of sodium azide maintained the concentration at 60% at four weeks. Neither fixative solution interfered with the radioimmunoassay except at very high concentrations. Fixation for 24h gave the highest estimates of each of the peptides. It is concluded that fixation can be a useful alternative to freezing for preservation of peptides in tissue for radioimmunoassay.     

The stability of prostaglandin E1 in dilute physiological solutions at 37 degrees C

The stability of prostaglandin E1 (PGE1) in three physiologic solutions was studied at body temperature (37 degrees C) over 32 days. The solutions were 100 mcg/ml PGE1 in isotonic saline (pH 4.5), 0.1 M phosphate buffered water (pH 7.4) or 0.01 M phosphate buffered isotonic saline (pH 4.7). PGE1 was found to be more stable in the saline and buffered saline solutions at the pH values of 4.5 and 4.7 respectively. Twenty-five per cent of the PGE1 remained at 32 days in these solutions while 95% of the PGE1 in the solution at pH 7.4 was degraded by day 14. The degradation of PGE1 in the acidic solutions appeared to be nearly linear when plotted on a semilog graph. This data allows one to use PGE1 in an aqueous, slightly acidic solution in a system that requires it to be kept at 37 degrees C for up to 30 days such as a biologically implantable pump. Investigators can use such a system in vivo to study the effect of known concentrations of PGE1 given over a period of time to a specific area of interest.     

A thin-film electrochemical study of the "blue" copper proteins,auracyanin A and auracyanin B,from the photosynthetic bacterium <Emphasis Type="Italic">Chloroflexus</Emphasis><Emphasis Type="Italic">aurantiacus</Emphasis>: the reduction potential as a function of pH

The reversible formal potentials of auracyanin A and auracyanin B, two closely related "blue" copper proteins from the photosynthetic bacterium Chloroflexus aurantiacus, have been determined by protein film voltammetry in the range 4相似文献   

Instability of rat liver chromatin and other nuclear non-histone proteins in alkaline solution.

Non-histone proteins from rat liver nuclei and chromatin were shown to be hydrolysed in 0.1M or-1M-NaOH solutions both at 4 degrees and 18 degrees C; 24h in 1M-NaOH at 18 degrees C is sufficient to break down approx. 77% of these proteins to low-molecular-weight peptides. Loss of protein material banding in the region of pH5.5-8.0 has been demonstrated by isoelectric focusing in polyacrylamide gels, and fine high-molecular-weight bands are no longer visible on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results indicate that care must be taken when analysing non-histone-protein fractions to avoid exposure to alkaline pH conditions.     

Use of proline-specific endopeptidase in the isolation of all four "native" disulfides of hen egg white lysozyme

The disulfide peptides from the tryptic digestion of cyanogen bromide-treated hen egg white lysozyme (HEWL) were isolated by reverse phase high performance liquid chromatography (HPLC) and identified by amino acid analysis. Three peptides containing the I-VIII, II-VII, and III-V + IV-VI disulfide bonds were obtained. The two-disulfide peptide was further digested with proline-specific endopeptidase (PCE) (EC 3.4.21.26). Amino acid analysis of digest peptides separated by HPLC showed four peptides with the IV-VI disulfide bond as well as a peptide with the III-V disulfide bond. The IV-VI peptides were produced by hydrolysis of several alanine-X bonds as well as the prolyl-cystine bond. Our studies show that alanyl peptide bonds to lysyl, seryl, and leucyl residues are susceptible to hydrolysis by PCE preparations, thus substantially extending its known specificity range. The two-disulfide peptide was also digested sequentially with thermolysin and PCE; the resulting IV-VI and III-V peptides were identified by HPLC and amino acid analysis. PCE showed substantial activity at pH 5.3 as well as at pH 8.3. The lower pH is useful in studies of proteins or peptides where base-catalyzed reactions must be limited.     

Androgen-dependent peptides of the rat ventral prostate nuclear envelope

Nuclear envelope peptides of the rat ventral prostate in the molecular weight region 18,400 to 19,400 show marked androgen dependence. After castration these peptides disappear. Re-administration of testosterone restores them. They were not extracted by 0.1 M NaCl or 5% HClO4 but were partially extracted by 0.35 M NaCl and 1% Triton. These peptides were not present in rat liver nuclear envelopes. As these androgen-dependent non-histone peptides have similar characteristics to androgen-dependent peptides identified in the prostate nucleus, we conclude that the nuclear peptides are at least partially localized to the nuclear envelope.     

Cry3Bb1 protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in root exudates and biomass of transgenic corn does not persist in soil

The Cry3Bb1 protein, insecticidal to the corn rootworm complex (Diabrotica spp.), of Bacillus thuringiensis (Bt) subsp. kumamotoensis was released in root exudates of transgenic Bt corn (event MON863) in sterile hydroponic culture (7.5 +/- 1.12 ng/ml after 28 days of growth) and in nonsterile soil throughout growth of the plants (2.2 +/- 0.62 ng/g after 63 days of growth). Kitchawan soil, which contains predominantly kaolinite (K) but not montmorillonite (M), was amended to 3 or 6% (vol./vol.) with K (3K and 6K soils) or M (3M and 6M soils) and with 1, 3, 5, or 10% (wt./wt.) of ground biomass of Bt corn expressing the Cry3Bb1 protein and incubated at 25 +/- 2 degrees C at the -33-kPa water tension for 60 days. Soils were analyzed for the presence of the protein every 7 to 10 days with a western blot assay (ImmunoStrip) and verified by ELISA. Persistence of the protein varied with the type and amount of clay mineral and the pH of the soils and increased as the concentration of K was increased but decreased as the concentration of M was increased. Persistence decreased when the pH of the K-amended soils was increased from ca. 5 to ca. 7 with CaCO(3): the protein was not detected after 14 and 21 days in the pH-adjusted 3K and 6K soils, respectively, whereas it was detected after 40 days in the 3K and 6K soils not adjusted to pH 7. The protein was detected for only 21 days in the 3M soil and for 14 days in the 6M soil, which were not adjusted in pH. These results indicate that the Cry3Bb1 protein does not persist or accumulate in soil and is degraded rapidly.     

A deacylation enzyme for aculeacin A, a neutral lipopeptide antibiotic, from Actinoplanes utahensis: purification and characterization

An enzyme, tentatively termed aculeacin A acylase, useful in preparing deacylated peptides which are used as starting material for semisynthetic antifungal antibiotics, was purified from the culture filtrate of Actinoplanes utahensis NRRL12052. The purification involved ultrafiltration and column chromatographies on DEAE-cellulose, hydroxyapatite, and Butyl-Toyopearl 650M. The purified enzyme was composed of two dissimilar subunits with molecular weights of 55,000 and 19,000. The subunits were dissociated in the presence of 0.1% SDS or 6 M guanidine hydrochloride; the dissociation accompanied loss of acylase activity. The enzyme was fully active at pH 7.0 and at 60 degrees C. Its pI was estimated to be above 10.25. The Km and Vmax for aculeacin A were 1.53 mM and 39.7 mumol/min/mg-protein, respectively.     

Acid hydrolases in HeLa cells: comparison of methods for light microscopy

To distinguish lysosome populations of HeLa cells, acid phosphatase, beta-glucuronidase, arylsulfatase and esterase were demonstrated using various substrates and couplers with different fixations, pHs and inhibitors. The substrates chosen were for acid phosphatase, naphthol AS-BI phosphate with fast red violet LB at pH 4.6; for beta-glucuronidase, naphthol AS-BI beta-D-glucuronide with fast red violet LB at pH 4.4; for arylsulfatase, p-nitrocatechol sulfate, with lead as the capturing ion, at pH 4.8 and 5.6; and for esterase, naphthol AS-D acetate with fast blue BB at pH 6.5. In the azo-dye methods, the coupling was always simultaneous and results were satisfactory with unfixed cells. For optimal demonstration of arylsulfatase, cells were fixed in glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, 2% for 24 hr or 6.25% for 2 hr, and washed for 1-9 days in 0.1 M veronal acetate buffer pH 7.2, 7.5% with respect to sucrose. Two groups of lysosomes were distinguished. One comprised small bodies, probably primary lysosomes, which lay in a cluster near the nucleus. They had quite stable membranes and were mostly acid phosphatase-positive. They sometimes contained beta-glucuronidase or esterase, but rarely arylsulfatase. The other group included all the acid hydrolase-positive bodies scattered throughout the rest of the cytoplasm. They were mostly larger, with more labile membranes, and contained beta-glucuronidase, esterase or arylsulfatase, but rarely acid phosphatase.     

Elution of antibodies from a Protein-A column by aqueous arginine solutions

Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatography.