Acyl and amino intermediates in penicillopepsin-catalysed reactions and activation by nonsubstrate peptides

1977 ◽  
Vol 55 (4) ◽  
pp. 286-294 ◽  
Author(s):  
Tusn. T. Wang ◽  
Theo Hofmann
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Acyl Transfer ◽  
High Yield ◽  
Major Conclusion ◽  
Small Peptides ◽  
Trypsinogen Activation ◽  
The Third ◽  
Effects Of Ph ◽  
Relative Specificity

The action of penicillopepsin on a variety of small peptides was studied qualitatively and kinetically. With the substrate benzyloxycarbonyl-Phe-Leu, benzyloxycarbonyl-Tyr-Leu and acetyl-Phe-Tyr the formation of Leu-Leu from the first two and Tyr-Tyr from the third substrate was observed. These reactions presumably proceed via a covalent amino-intermediate in analogy to pig pepsin. However, with penicillopepsin the yields of transpeptidation are low. In contrast, transpeptidations via acyl transfer proceed in high yield with a variety of substrates. With Leu-Trp-Met both acyl and amino transfer occurs as shown by the formation of Leu-Leu and Met-Met; unlike with pig pepsin, however, no Leu-Leu-Leu or Met-Met-Met was observed. Nonsubstrate peptides activate the cleavage of small substrates. (The term 'cleavage' is used here to imply that the reaction is either a hydrolysis or a transpeptidation, or both. The term 'hydrolysis' will only be used for strictly hydrolytic reactions.) As with pig pepsin the activators increase predominantly the transpeptidation reaction and have only small effects on hydrolysis. The activators increase kcat but have no effect on Km. Studies of the cleavage of six different peptides show that at pH 4.7 Km is lower than at pH 3.4 while kcat is unaffected. As with pig pepsin activation by nonsubstrate peptides is greater at pH 4.7 than at pH 3.4. Benzyloxycarbonyl-Glu-Tyr which is an inhibitor of trypsinogen activation (Ki = 50 μM) is an activator for the cleavage of Leu-Tyr-NH2 (Ka = 500 μM). Pepstatin, an inhibitor of the proteolytic activity of acid proteases, also inhibits the action of penicillopepsin on Leu-Tyr-NH2.The major conclusion from these studies is that the action of penicillopepsin on small peptides is qualitatively similar to that of pig pepsin. Transpeptidation reactions of both the amino acid and the acyl transfer type have been observed. However, there are considerable differences in the effects of pH, and in relative specificity between the two enzymes.

scholarly journals Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products

1976 ◽  
Vol 153 (3) ◽  
pp. 691-699 ◽  
Author(s):  
T T Wang ◽  
T Hofmann
Keyword(s):  
Reaction Rates ◽  
Major Product ◽  
Conclusive Evidence ◽  
Acyl Transfer ◽  
High Yield ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
Ph Optimum ◽  
Covalent Intermediate ◽  
Hydrolysis Of

The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.

BOTANICAL COMPOSITION AND PRODUCTIVITY OF PERENNIAL GRASSES AGROPHYTOCENOSES WITH TETRAPLOID MEADOW CLOVER

1970 ◽  
pp. 59-62
Author(s):  
N. I. Kasatkina ◽  
Zh. S. Nelyubina
Keyword(s):  
Dry Weight ◽  
Single Species ◽  
Lotus Corniculatus ◽  
Specific Weight ◽  
Biological Properties ◽  
Phleum Pratense ◽  
Perennial Grasses ◽  
High Yield ◽  
Botanical Composition ◽  
The Third

The biological properties of plants, their mutual relations under different growth conditions and at different periods of their life, must be known for obtaining highly productive agrophytocenoses with participation of a meadow clover (Trifolium pratense L.). Botanical composition and fodder productivity of perennial grasses in agrocenoses with participation of meadow tetraploid clover Kudesnik were studied in 2014-2017. It was revealed that in the first and second years of use the agrophytocenosis, the yield of green mass was formed due to meadow tetraploid clover, the share of its participation in the first mowing was at level of 71-87% and 64-97% respectively. Specific weight of clover in multispecies agrocenoses considerably decreased by the third year of use: in the first mowing up to 32-68%, in the second - up to 8-52%. At the same time, the percentage of long-term herbaceous grasses increased: meadow timothy (Phleum pratense L.) - up to 34-54%, eastern galega (Galéga orientális Lam.) - up to 33%, changeable alfalfa (Medicago x varia Martyn) - up to 22-54%, lotus corniculatus (Lotus corniculatus L.) - up to 14-19%. The proportion of weed admixture in single-species clover planting was 12%, in agrocenoses - 2-14%. The grass mixtures clover + timothy and clover + alfalfa + timothy were less infested by weeds. High yield of dry weight of single-species sowing of meadow tetraploid clover was obtained in the first two years of use - 7.8 and 6.5 tons / ha, respectively. By the third year of use, the productivity of clover has decreased to 2.9 t / ha. On average, for three years of use, the highest yield (6.2-6.3 t / ha) was formed by agrocenoses meadow tetraploid clover + meadow timothy and meadow tetraploid clover + changeable alfalfa + meadow timothy.

scholarly journals FURTHER STUDIES ON THE ROLE OF PROTEASES IN THE ALLERGIC REACTION

1961 ◽  
Vol 113 (2) ◽  
pp. 359-380 ◽  
Author(s):  
Georges Ungar ◽  
Takuso Yamura ◽  
Jacqueline B. Isola ◽  
Sidney Kobrin
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Proteolytic Activity ◽  
Guinea Pigs ◽  
Protease Activity ◽  
Amino Acid Esters ◽  
Protein Substrates ◽  
Protease Activation ◽  
Acid Esters

Protease activity was measured through the hydrolysis of synthetic amino acid esters in body fluids and tissues of guinea pigs, rats, mice, and humans. Significant in vitro activation was observed in serum and lung slices of sensitized guinea pigs on addition of the specific antigen. Increased proteolytic activity was also seen in reverse anaphylaxis. More marked activation occurred when guinea pig serum was treated with peptone and guinea pig or rat serum was treated with agar. Protease activation was demonstrated in specimens of human skin under the influence of a poison ivy extract or croton oil added in vitro. Urinary protease activity of guinea pigs increased significantly during the first hours of anaphylactic shock and very markedly in peptone shock. Peptone shock, elicited in mice pretreated with H. pertussis, was accompanied by a considerable increase in protease activity in the peritoneal fluid as compared with non-pretreated mice which were insensitive to peptone. Proteolytic activity resulting from the activation procedures was due to a number of proteases. The dominant substrate affinity and inhibition patterns suggest that serum and urine proteases are similar to but not identical with plasmin. Anaphylactic activation exhibited patterns different from those resulting from the action of anaphylactoid agents. Tissue enzymes are either of cathepsin- or chymotrypsin-type or mixtures of both. Some of the activated enzymes, although remarkably effective in hydrolyzing amino acid esters, show no activity on protein substrates. This does not justify, however, their designation as "esterases." They probably belong to the class of specific proteases acting only on a single or a small number of functionally significant protein substrates. There is at present sufficient evidence to prove not only that protease activation does occur in anaphylaxis and anaphylactoid conditions but also that it is an important component of the chain of reactions leading to the allergic response.

scholarly journals Point Mutations in Transmembrane Helices 2 and 3 of ExbB and TolQ Affect Their Activities in Escherichia coli K-12

2004 ◽  
Vol 186 (13) ◽  
pp. 4402-4406 ◽  
Author(s):  
Volkmar Braun ◽  
Christina Herrmann
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Transmembrane Helices ◽  
The Third ◽  
Form Part ◽  
K 12

ABSTRACT Replacement of glutamate 176, the only charged amino acid in the third transmembrane helix of ExbB, with alanine (E176A) abolished ExbB activity in all determined ExbB-dependent functions of Escherichia coli. Combination of the mutations T148A in the second transmembrane helix and T181A in the third transmembrane helix, proposed to form part of a proton pathway through ExbB, also resulted in inactive ExbB. E176 and T148 are strictly conserved in ExbB and TolQ proteins, and T181 is almost strictly conserved in ExbB, TolQ, and MotA.

scholarly journals The effect of feeding high levels of low-quality proteins to growing chickens

1975 ◽  
Vol 34 (3) ◽  
pp. 363-373 ◽  
Author(s):  
E. Wetnli ◽  
T. R. Morris ◽  
T. P. Shresta
Keyword(s):  
Growth Rate ◽  
Amino Acid ◽  
Dietary Protein ◽  
Soya Bean ◽  
Maximum Efficiency ◽  
Food Utilization ◽  
The Third ◽  
Bean Meal ◽  
Groundnut Meal ◽  
Soya Bean Meal

1. Three growth trials were done using male broiler chicks. In the first two trials, groundnut meal was used, with and without supplementary methionine and lysine. In the third trial, soya-bean meal was used with and without supplementary methionine. Protein levels ranged in the first trial from 120 to 420 g/kg diet and in the third trial from 120 to 300 g/kg diet. Thus the assumed minimal amino acid requirements of the chick were supplied by high levels of low-quality dietary protein.2. Diets based on cereals and groundnut meal did not support maximum live-weight gain or maximum efficiency of food utilization at any level of dietary protein. When the principal deficiencies of lysine and methionine were corrected, this protein mixture was capable of supporting the same growth rate as a control diet of cereals and herring meal.3. Diets based on maize and soya-bean meal did not support quite the same growth rate as similar diets supplemented with methionine, even though the protein level in the unsupplemented diets was sufficient to meet the assumed methionine requirements.4. These results are interpreted as examples of amino acid imbalance in diets composed of familiar feeding-stuffs. It is concluded that one cannot assume that the poor quality of a protein source can always be offset by increasing the concentration of dietary protein.

Stabilizing unusual conformations in small peptides and glucopeptides using a hydroxylated cyclobutane amino acid

2009 ◽  
Vol 7 (14) ◽  
pp. 2885 ◽  
Author(s):  
Alberto Fernández-Tejada ◽  
Francisco Corzana ◽  
Jesús H. Busto ◽  
Alberto Avenoza ◽  
Jesús M. Peregrina
Keyword(s):  
Amino Acid ◽  
Small Peptides

Aminostratigraphic Correlation and Geochronology of Two Quaternary Loess Localities, Central Mississippi Valley

1994 ◽  
Vol 41 (3) ◽  
pp. 289-297 ◽  
Author(s):  
June E. Mirecki ◽  
Barry B. Miller
Keyword(s):  
Amino Acid ◽  
Kinetic Model ◽  
Mississippi Valley ◽  
River Section ◽  
Mollusc Shell ◽  
The Third ◽  
Shell Carbonate ◽  
Parabolic Kinetic

AbstractAmino acid epimeric (aIle/Ile) values from terrestrial molluscs are used to define and correlate three aminozones in loess sequences exposed across the central Mississippi Valley, in Arkansas and Tennessee. Three superposed aminozones are defined at Wittsburg quarry, Arkansas, primarily using aIle/Ile values from total hydrolysates of the gastropod genus Hendersonia: Peoria Loess (aIle/Ile = 0.07 ± 0.01), Roxana Silt (0.14 ± 0.02), and a third loess (0.28 ± 0.06). Loess units at Wittsburg quarry can be correlated on lithologic characteristics eastward across the Mississippi Valley to the Old River section, near Memphis, Tennessee; however, only one loess unit is fossil-bearing (Peoria Loess, aIle/Ile = 0.05) at that section. Radiocarbon analyses of charcoal from the upper Roxana Silt (ca. 26,000 to 29,000 yr old) and mollusc shell carbonate from the basal Roxana Silt (ca. 39,000 yr old) are used to calibrate amino acid epimeric data for the central Mississippi Valley. These data, applied to the apparent parabolic kinetic model of R. M. Mitterer and N. Kriausakul (1989, Quaternary Science Reviews 8, 353-357), suggest an Illinoian (>120,000 yr) age for the third loess in the central Mississippi Valley that is correlative with part of the Loveland Loess in Illinois and Iowa.

scholarly journals Functional Characterization of the Proteolytic System of Lactobacillus sanfranciscensis DSM 20451T during Growth in Sourdough

2005 ◽  
Vol 71 (10) ◽  
pp. 6260-6266 ◽  
Author(s):  
Nicoline Vermeulen ◽  
Melanie Pavlovic ◽  
Matthias A. Ehrmann ◽  
Michael G. Gänzle ◽  
Rudi F. Vogel
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Peptide Transport ◽  
Transport Systems ◽  
Peptide Hydrolysis ◽  
Degenerate Primers ◽  
Lactobacillus Sanfranciscensis ◽  
Peptide Uptake

ABSTRACT Protein hydrolysis and amino acid metabolism contribute to the beneficial effects of sourdough fermentation on bread quality. In this work, genes of Lactobacillus sanfranciscensis strain DSM 20451 involved in peptide uptake and hydrolysis were identified and their expression during growth in sourdough was determined. Screening of the L. sanfranciscensis genome with degenerate primers targeting prt and analysis of proteolytic activity in vitro provided no indication for proteolytic activity. Proteolysis in aseptic doughs and sourdoughs fermented with L. sanfranciscensis was inhibited upon the addition of an aspartic protease inhibitor. These results indicate that proteolysis was not linked to the presence of L. sanfranciscensis DSM 20451 and that this strain does not harbor a proteinase. Genes encoding the peptide transport systems Opp and DtpT and the intracellular peptidases PepT, PepR, PepC, PepN, and PepX were identified. Both peptide uptake systems and the genes pepN, pepX, pepC, and pepT were expressed by L. sanfranciscensis growing exponentially in sourdough, whereas pepX was not transcribed. The regulation of the expression of Opp, DtpT, and PepT during growth of L. sanfranciscensis in sourdough was investigated. Expression of Opp and DtpT was reduced approximately 17-fold when the peptide supply in dough was increased. The expression of PepT was dependent on the peptide supply to a lesser extent. Thus, the accumulation of amino nitrogen by L. sanfranciscensis in dough is attributable to peptide hydrolysis rather than proteolysis and amino acid metabolism by L. sanfranciscensis during growth in sourdough is limited by the peptide availability.

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