Trichloroacetic Acid-soluble Peptides Research Articles

Combination effect of phosphate and modified atmosphere on quality and shelf-life extension of refrigerated seabass slices

Effect of pretreatment with three different phosphate compounds including trisodium phosphate (TSP), sodium pyrophosphate (PP) and sodium tripolyphosphate (STPP) in combination with modified atmosphere packaging (MAP) (80%CO 2, 10%O 2, 10%N 2) on seabass slices stored at 4 °C was investigated. Phosphate pretreatment showed the synergistic effect with MAP on reduction of microbiological, chemical and sensory deterioration of seabass slices as evidenced by the lowered microbial counts, total volatile base and trimethylamine, compared with samples stored in air and those kept under MAP. Pretreatment with PP resulted in the retarded protein denaturation as evidenced by the reduced changes in sulfhydryl content and surface hydrophobicity during the extended storage. Increase in water uptake ability accompanied by the decreased exudate loss was observed in samples pretreated with phosphates, especially pyrophosphate. No marked autolytic degradation in samples kept under MAP with and without phosphate pretreatment was observed throughout the storage as indicated by no changes in trichloroacetic acid soluble peptide. Therefore, the effective retardation of microbiological, chemical, and sensory deterioration of seabass slices stored under MAP could be achieved by pretreatment with pyrophosphate.

Salt-induced structural changes in Mozzarella cheese and the impact upon free oil formation in ripening cheese

Mozzarella cheese was manufactured to different salt levels by a combination of dry- and brine-salting. Free oil formation was lower, apparent viscosity of melted cheese was higher, and expressible serum was lower for cheese at the higher salt levels. Salt had no effect on the extent of primary proteolysis as measured by pH 4.6 sodium acetate buffer or 120 g·L -1 trichloroacetic acid soluble peptides. Fat globule size decreased in the cheese after 20 d of ripening. Additional Mozza- rella curd was salted separately in a brine bath without additional dry-salting. At the centre of the brined block of curd where the salt content was lowest, free oil was highest and apparent viscosity was lowest. These results are discussed in relation to how salt affects free oil formation by altering the protein and water phases during ripening. Salt / mozzarella / cheese / fat globule / free oil

Some characteristics of commercial Som-fug produced in Thailand

Seven commercial brands of Som-fug were analysed for chemical composition, biogenic amines and acceptance. Commercial Som-fug contained 11.4–16.2% protein, 1.13–1.56% lipid, 69.66–77.08% moisture, 2.45–3.60% ash and 3.37–4.90% salt. The pH values ranged from 4.56 to 4.60 and total acidity varied from 1.42% to 2.35%. Lactic acid was the major organic acid in all samples, but citric acid, succinic acid and pyruvic acid were found only in some brands. Differences in the trichloroacetic acid soluble peptides and lipid oxidation, among all samples, were observed. The proteolysis of myosin heavy chain was observed on SDS–PAGE analysis. Additionally, the formation of biogenic amines, including tryptamine, putrescine, cadaverine, histamine and tyramine, was observed in commercial Som-fug, but β-phenylethylamine was not detectable in any samples. Different Som-fug samples showed different acceptabilities, especially for taste and appearance. Therefore, raw materials, ingredients and process should be factors for determining the characteristics of Som-fug products.

ATPASE ACTIVITY, SURFACE HYDROPHOBICITY, SULFHYDRYL CONTENT AND PROTEIN DEGRADATION IN REFRIGERATED SEABASS MUSCLE IN MODIFIED ATMOSPHERE PACKAGING

The effect of modified atmosphere packaging (80% CO2, 10% O2, 10% N2) on ATPase activity, surface hydrophobicity, sulfhydryl content and degradation of proteins in seabass muscle during storage at 4C was investigated. No changes in Ca2+-, Mg2+-, Mg2+-Ca2+-ATPase activities of natural actomyosin (NAM) in seabass slices kept under MAP were observed throughout the storage for up to 21 days (P > 0.05). However, a slightly increased Mg2+-EGTA-ATPase was found. For seabass slices stored under air atmosphere, Ca2+-ATPase activity decreased, whereas Mg2+-EGTA-ATPase activity increased (P < 0.05) with a concomitant loss in Ca2+-sensitivity. Lower decreases in total sulfhydryl content but higher increases in surface hydrophobicity were observed in samples stored under MAP, compared to those kept under air atmosphere. No marked autolytic degradation in samples kept under MAP was observed throughout the storage as monitored by no changes in myosin heavy chain, free α-amino acid and trichloroacetic acid soluble peptide. Conversely, a considerable degradation was found in samples kept under air atmosphere, especially after 9 days of storage. Therefore, MAP is a promising means to retard the changes in muscle proteins, especially degradation.

Processing of chromogranin A by plasmin provides a novel mechanism for regulating catecholamine secretion.

Chromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is also distributed widely in secretory vesicles throughout the neuroendocrine system. CgA contains the sequences for peptides that modulate catecholamine release, but the proteases responsible for the release of these bioactive peptides from CgA have not been established. We show here that the major fibrinolytic enzyme, plasmin, can cleave CgA to form a series of large fragments as well as small trichloroacetic acid-soluble peptides. Peptides generated by plasmin-mediated cleavage of CgA significantly inhibited nicotinic cholinergic stimulation of catecholamine release from PC12 cells and primary bovine adrenal chromaffin cells. We also show that the zymogen, plasminogen, as well as tissue plasminogen activator bind saturably and with high capacity to catecholaminergic (PC12) cells. Occupancy of cell surface binding sites promoted the cleavage of CgA by plasmin. Positive and negative modulation of the local cellular fibrinolytic system resulted in substantial alterations in catecholamine release. These results suggest that catecholaminergic cells express binding sites that localize fibrinolytic molecules on their surfaces to promote plasminogen activation and proteolytic processing of CgA in the environment into which CgA is secreted to generate peptides which may regulate neuroendocrine secretion. Interactions between CgA and plasmin(ogen) define a previously unrecognized autocrine/paracrine system that may have a dramatic impact upon catecholamine secretion.

Comparative analysis of the kinetic parameters and thermal stability of two matrix metalloproteinases expressed in the developing sea urchin embryo

The extracellular matrix is now recognized as a dynamic structure which influences cellular properties. Many matrix metalloproteinase activities have been identified and characterized in vertebrates and constitute important agents in controlling the composition of the extracellular matrix. We have begun a study of matrix metalloproteinase activities in the developing sea urchin embryo. Using sea urchin peristome collagen or gelatin as physiological substrates we have determined the kinetic parameters, K m and V max, for an 87 kDa gelatinase activity expressed in late stage sea urchin embryos. We also determined the kinetic parameters K m, V max and k cat, for a 41 kDa species, expressed in the early sea urchin embryo, which possesses both collagenase and gelatinase activities. All values determined were similar to those reported in the literature for vertebrate collagenases and gelatinases and K m values in the micromolar range suggest that both species possess physiologically relevant activities. Both activities have previously been shown to require Ca 2+ for activity. Using an assay for quantitating the cleavage of gelatin into trichloroacetic acid soluble peptides we report here markedly different effects of Ca 2+ on the thermal denaturation profiles of the gelatinases. This latter finding may be indicative of different modes of action for this activating cation. Collectively, these results demonstrate both similarities and differences between vertebrate and invertebrate sea urchin gelatinases.

Peptide mapping of β-casein by capillary zone electrophoresis

A method to the study of β-casein proteolysis by aspartic proteinases is developed. The 3% trichloroacetic acid-soluble peptides of β-casein digested with cardosin A were separated by capillary zone electrophoresis under different experimental conditions in an uncoated fused silica capillary. The best separation was at pH 3.01, 30 kV and 30 °C.

Enumeration of amino acid fermenting bacteria in the human large intestine: effects of pH and starch on peptide metabolism and dissimilation of amino acids

Proteins and trichloroacetic acid-soluble peptides were present in high concentrations in human intestinal contents and faeces. Free amino acids were also detected in millimolar amounts in proximal and distal colon contents, with hydroxyproline, alanine, lysine and valine predominating, showing that a wide variety of organic N-containing compounds was available for fermentation by intestinal bacteria. Measurements of products of dissimilatory amino acid metabolism (ammonia, branched chain fatty acids) demonstrated that these substances occurred in all regions of the large bowel. Amino acid fermenting populations were enumerated in faeces obtained from five healthy donors by most probable number analysis. Counts ranged from 10 10 to 10 11 per gram dry weight faeces. Acetate, propionate and butyrate were the principal fermentation acids in the most probable number tubes. Bacteria forming branched chain fatty acids as major end products of metabolism ranged from 0.6% (isovalerate/2-methylbutyrate) to 40% (isobutyrate) of total peptide and amino fermenting populations. Plate counts also gave high values for peptide fermenting communities in the region of 10 11 per gram dry weight faeces, though considerably lower numbers of organisms grew on plates containing either single amino acids or Stickland pairs. Clostridia and anaerobic Gram-positive cocci were the predominant isolates in these studies. Physiological investigations on the effects of pH and carbohydrate availability on peptide and amino acid fermentation by intestinal bacteria showed that two environmental characteristics of the proximal colon (low pH, high carbohydrate availability), reduced the rate and net ammonia production from peptides, while carbohydrate (starch) was more important in this respect in amino acid fermentation vessels. Starch reduced initial rates of production of branched chain fatty acids by approximately 35% in peptide fermentations, however, culture pH was a more significant determinant affecting formation of these metabolites. Comparisons of branched chain fatty acid formation by faecal bacteria at pH 6.8 and 5.5 showed that their production was reduced by over 60% in pH 5.5 cultures. These data demonstrate that by increasing bacterial requirements for organic N-containing compounds for use in biosynthetic reactions, and through fermentation acid production, carbohydrate availability plays a major role in regulating dissimilatory metabolism of peptides and amino acids in the human large intestine.

Enumeration of amino acid fermenting bacteria in the human large intestine: effects of pH and starch on peptide metabolism and dissimilation of amino acids

Proteins and trichloroacetic acid-soluble peptides were present in high concentrations in human intestinal contents and faeces. Free amino acids were also detected in millimolar amounts in proximal and distal colon contents, with hydroxyproline, alanine, lysine and valine predominating, showing that a wide variety of organic N-containing compounds was available for fermentation by intestinal bacteria. Measurements of products of dissimilatory amino acid metabolism (ammonia, branched chain fatty acids) demonstrated that these substances occurred in all regions of the large bowel. Amino acid fermenting populations were enumerated in faeces obtained from five healthy donors by most probable number analysis. Counts ranged from 1010 to 1011 per gram dry weight faeces. Acetate, propionate and butyrate were the principal fermentation acids in the most probable number tubes. Bacteria forming branched chain fatty acids as major end products of metabolism ranged from 0.6% (isovalerate/2-methylbutyrate) to 40% (isobutyrate) of total peptide and amino fermenting populations. Plate counts also gave high values for peptide fermenting communities in the region of 1011 per gram dry weight faeces, though considerably lower numbers of organisms grew on plates containing either single amino acids or Stickland pairs. Clostridia and anaerobic Gram-positive cocci were the predominant isolates in these studies. Physiological investigations on the effects of pH and carbohydrate availability on peptide and amino acid fermentation by intestinal bacteria showed that two environmental characteristics of the proximal colon (low pH, high carbohydrate availability), reduced the rate and net ammonia production from peptides, while carbohydrate (starch) was more important in this respect in amino acid fermentation vessels. Starch reduced initial rates of production of branched chain fatty acids by approximately 35% in peptide fermentations, however, culture pH was a more significant determinant affecting formation of these metabolites. Comparisons of branched chain fatty acid formation by faecal bacteria at pH 6.8 and 5.5 showed that their production was reduced by over 60% in pH 5.5 cultures. These data demonstrate that by increasing bacterial requirements for organic N-containing compounds for use in biosynthetic reactions, and through fermentation acid production, carbohydrate availability plays a major role in regulating dissimilatory metabolism of peptides and amino acids in the human large intestine.

Heat-induced hydrolysis of sodium caseinate

Abstract The formation of pH 4.6- and 2% trichloroacetic acid-soluble peptides from sodium caseinate solutions (2.5%, w v ) on heating at 140 °C in the pH range 1–11 was studied using alkaline urea-polyacrylamide gel electrophoresis (urea-PAGE) and reversed phase (RP)-HPLC. The pH of the caseinate solution had marked qualitative and quantitative effects on the soluble peptides produced on heating. Soluble peptides were formed linearly on heating at 140 °C and pH 7.0 for 100 min. Fifteen of the pH 4.6-soluble peptides produced at pH 7.0 were isolated and identified: 9 peptides originated from αs1-casein (5 from the C-terminal region), 1 from αs2-casein, 3 from β-casein and 2 from κ-casein.

Phospholipase activation in the cytotoxic mechanism of thionin purified from nuts of Pyrularia pubera

Treating NIH3T3 fibroblast cells with Pyrularia pubera thionin (100 micrograms/ml) stimulated release of labelled free fatty acids from phospholipids biosynthetically labelled by incorporation of [3H]arachidonic acid. Since Pyrularia thionin exhibited no detectable phospholipase activity, it was concluded that the release response represented activation of endogenous phospholipases in the cells. The phospholipase activated by Pyrularia thionin (100 micrograms/ml) stimulated the release of 61% of the incorporated [3H]arachidonate in the presence of 1.8 mM extracellular calcium with maximum activation at 90 min following an initial lag period of about 20 min. The release response exhibited little dependence on extracellular calcium at this thionin concentration, but at concentrations 20 micrograms/ml, extracellular calcium appeared to be inhibitory to phospholipase activation. Some characteristics of the fatty acid release response are consistent with activation of a lysosomal phospholipase being part of the cellular response to Pyrularia thionin. Activation of a lysosomal enzyme can occur independently or as a result of coordinate activation of the whole lysosome, which would expose other cellular components of degradative lysosomal enzymes. Consistent with coordinate activation of lysosomal enzymes, Pyrularia thionin also stimulates the production of small, trichloroacetic acid-soluble peptides and nucleic acid fragments from biosynthetically-labelled RNA and proteins in treated cells. It is not clear from the results obtained what role, if any, activation of lysosomal enzymes plays in the overall toxic response to Pyrularia thionin in NIH3T3 cells. However, Pyrularia pubera thionin may represent a useful tool for studying the regulation of lysosomal enzymes and their roles in cells.

Limited proteolysis of 3-phosphoglycerate kinase without loss of enzymic activity

During tryptic digestion of pig muscle 3-phosphoglycerate kinase in the presence of 3-phosphoglycerate both the decrease of enzymic activity and the release of trichloroacetic acid-soluble peptides occur after a pronounced lag period. During this lag phase the native enzyme molecule is split into two fragments with molecular masses of about 30 and 18 kDa, as detected by SDS-PAGE. Under non-denaturing conditions, however, these fragments are held together by non-cova-lent forces and constitute an active, nicked enzyme molecule. In the absence of substrates or in the presence of MgATP the kinetics of tryptic digestion is apparently a single first order reaction leading to the formation of peptides with molecular masses of less than 10 kDa.

Protease activities in cultured beef lens epithelial cells peak and then decline upon progressive passage

Beef lens cells in culture are readily obtained and provide many opportunities to study phenomena related to cell differentiation and maturation, environmental stress, disease, and perhaps mechanisms of transformation. Although altered rates of proteolysis are known to accompany these phenomena, the proteolytic activities available in cultured beef lens epithelial cells have not been documented. In this work are documented the specific activities, based on protein and DNA content, of neutral exo- and endopeptidase, cathepsins B- and D-like enzymes and acid phosphatase in lens epithelial cortical and core tissue and in cultured epithelial cells at passages 1-43. Maximal activity of each protease occurs almost routinely at passage 5 or 9, reaching values of approx. 1400-, 0.77-, 4520-nmol min-1 per mg protein for neutral exopeptidase (passage 5), neutral endopeptidase (passage 5) and cathepsin B (passage 5) respectively, and 7.1 micrograms trichloroacetic acid soluble peptide min-1 per mg protein for cathepsin D (passage 15). On a microgram-1 DNA basis, the maximal specific activities for the same enzymes were 48 (passage 5), 0.03 (passage 5), 283 (passage 9), and 0.5 (passage 9) respectively. In subsequent passages, the specific activities declined to values which were similar to or lower than the specific activities observed for these proteases in lens epithelial tissue.

Relative Tryptic Digestion Rates of Food Proteins

ABSTRACTThe kinetics of tryptic digestion of different food proteins were studied by measuring trichloroacetic acid soluble peptide release and peptide bond splitting either by proton titration at constant pH or by recording the pH drop in nonbuffered suspensions. The theoretical basis of the pH drop assay was described. Application of this method for comparative studies requires complementary determinations of buffering capacities of the samples and the time course of the reference protein digestion. It was shown that milk powder preparations differed in digestion rates by a factor of two and various lots of commercial soy meals differed as much as by a factor of three. Relative digestion rates of some food proteins from different sources could not be characterized by a single figure.

A sensitive fluorometric assay for the simultaneous estimation of pepsin and pepsinogen in gastric mucosa

An assay for pepsin has been developed based on the fluorometric measurement of trichloroacetic acid-soluble peptides released from casein at pH 5.3. The increase in relative fluorescence was most sensitive in the range 10–50 μg pepsin/1 and casein hydrolysis was not affected by the addition of up to a 1000-fold molar excess of pepsinogen. This assay has been used to measure the free and total acid proteinase content of biopsies (< 5 mg) from different areas of the gastric mucosa of rat and man. Interference by the major lysosomal acid hydrolase. cathepsin D, could be eliminated by the differential stability of pepsin and cathepsin D at acid and neutral pH. The free acid proteinase activity of biopsies from the corpus were almost identical in these species whereas the total acid proteinase activity was approximately 5-fold greater in man.

Cathepsin D catalyzed degradation of cardiac mitochondria. A study of enzyme inactivation and ultrastructure

The ability of cathepsin D to catalyze degradation of intact mitochondria in vitro was used as a probe to investigate the possible role of this protease in the initiation of ischemia-induced mitochondrial injury. Cathepsin D mediated protein degradation was monitored using: 1. (1) Degradation of gross mitochondrial protein as assessed by changes in trichloroacetic acid soluble peptide formation. 2. (2) Inactivation of specific mitochondrial enzymes vis monoamine oxidase (MAO), adenylate kinase (AK), creatine kinase (CK), Ca 2+-Mg 2+ adenosine triphosphatase (ATPase) and malate dehydrogenase (MDH) in intact and solubilized mitochondrial preparations. 3. (3) Electron microscopy. The pH-activity profile for cathepsin D mediated mitochondrial protein degradation revealed an optimum at pH 3.2 to 3.4 with 12 and 5% optimal activity remaining at pH values of 5.5 and 6.0 respectively. Incubation of intact mitochondria with cathepsin D (pH 5.7 at 37°C) revealed that MAO was inactivated to a small extent (4.5% loss in activity following a 60-min incubation) while other enzymes were unaffected. In similar experiments with solubilized mitochondria however, MAO, AK and ATPase were moderately inactivated (10, 20 and 15% at 60 min respectively) while CK and MDH were unaffected. Pepstatin A was found to inhibit the observed inactivation indicating that cathepsin D and not a contaminating protease was responsible for the observed effects. Results from electron microscopy, paralleled those of the incubation studies indicating that there was little effect of cathepsin D on the intact organelle. The results suggest that while mitochondria contain proteins which are susceptible to cathepsin D catalyzed hydrolysis, the accessibility of cathepsin D to these proteins is limited in intact mitochondria. If cathepsin D is responsible for either the physiologic turnover of mitochondria or the mitochondrial destruction characteristic of ischemic injury in myocardial tissue, a concomitant disruption of the mitochondrial membrane by other groups of lysosomal or non-lysosomal enzymes must be postulated.

Analytical subcellular fractionation of rat pituitary homogenates, with special reference to prolactin proteolysis by lysosomes

Prolactin proteolysis by rat pituitary homogenates was assayed by measuring the release of trichloroacetic acid-soluble peptides from 125I-labelled rat prolactin. There was a distinct optimum at pH 4.3, with only trace amounts of activity at neutral and alkaline pH. Rat pituitary homogenates were subjected to analytical subcellular fractionation by sucrose density gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their respective marker enzymes, including: cytosol (lactate dehydrogenase); plasma membrane (5′-nucleotides); lysosomes ( N-acetyl- β-glucosaminidase, β-glucuronidase); mitochondria (particulate malate dehydrogenase); endoplasmic reticulum (neutral α-glucosidase); prolactin granules (radioimmunoassayable prolactin). Acid prolactin protease had a similar distribution to the lysosomal marker enzymes. A localisation of the activity to lysosomes was confirmed by subcellular fractionation experiments in which the lysosomes were selectively disrupted with low concentrations of the membrane pertubant, digitonin. Experiments with specific inhibitors of the lysosomal cathepsins indicate that both cathepsins B and D are implicated in pituitary prolactin proteolysis.

Proteases of Cerastes cerastes (Egyptian sand viper) and Cerastes vipera (Sahara sand viper) snake venoms

Proteases from Cerastes cerastes and Cerastes vipera venoms digested casein, gelatin and hemoglobin. Gelatin digestion was detected by reacting the trichloroacetic acid soluble peptides with trinitrobenzene sulfonate. This method is more sensitive than ultraviolet absorption, and is applicable to protein substrates irrespective of their aromatic amino acid content. The proteases showed optimal pH values of 7·8 for casein and 7·0 for hemoglobin digestion. They were activated by Ca 2+, Ba 2+ and Mg 2+, and were completely inhibited by 1 mM ethylenediamine tetra-acetate. This inhibition could be reversed maximally (73%) by 3 mM Ca 2+. Electrophoresis at pH 6·0 resolved the proteases into a major band migrating very slowly towards the cathode (3 mm/hr) and a minor more rapidly migrating band (14 mm/hr). Sephadex G—100 gel filtration resolved the proteolytic activity into 4 peaks in each venom. Their estimated mol. wts were 120,000, 49,000, 23,000 and 16,000 in C. cerastes and 130,000, 56,000, 25,000 and 14,000 in C. vipera. The first peak in both venoms showed the highest protease activity and was also the most lethal, causing marked hemorrhages in mice.

Degradation of intracellular protein in Salmonella typhimurium peptidase mutants

Multiply peptidase-deficient mutant strains of Salmonella typhimurium fail to carry out normal protein degradation during starvation for a carbon source. In these mutants, the extent of protein breakdown during starvation is about fourfold less than in the wild type. The products of protein breakdown in the mutant are mainly small, trichloroacetic acid-soluble peptides, not free amino acids as in the wild type. The carbon-starved mutant strain produces only about one thirtieth as much free amino acid from protein as the wild type. As a result, protein synthesis during starvation is reduced in the mutant compared to the wild type and the mutant strain shows a greatly prolonged lag phase after a nutritional shift-down.

Peptide accumulation during growth of peptidase deficient mutants

Mutant strains of Salmonella typhimurium simultaneously lacking peptidases N, A, B and D accumulate a heterogeneous mixture of small, trichloroacetic acid-soluble peptides during growth in minimal medium. Approximately 20% of the labelled leucine supplied to a growing culture of the mutant strain is converted to peptides. These peptides accumulate inside the cells before being released into the growth medium. Although the origin of these peptides has not been established, there are several processes that might contribute peptides to this pool. These include (1) turnover of signal sequences, (2) turnover of attenuator peptides, and (3) degradation of prematurely terminated proteins. These results indicate that the same family of peptidases that catabolizes exogenously supplied peptides and functions in carbon-starvation-induced protein turnover also hydrolyzes peptides generated during normal exponential growth.