Proteinases in normal bovine milk and their action on caseins

1983 ◽  
Vol 50 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Anthony T. Andrews
Keyword(s):  
Trypsin Inhibitor ◽  
Intermediate Product ◽  
Proteolytic Enzymes ◽  
Bovine Milk ◽  
Raw Milk ◽  
Soya Bean ◽  
Protein Breakdown ◽  
Casein Hydrolysis ◽  
Proteose Peptone ◽  
Bean Trypsin Inhibitor

SummaryNative proteolytic enzymes in good quality normal bovine milk readily hydrolyscd the caseins during incubation or storage, producing the γ-caseins, proteosc-peptone components 5 (PP5) and 8-fast (PP8F) and a considerable number of other unidentified fragments, many of which were also subsequently found in the proteose-pcptone fraction. The rate of casein hydrolysis was greater in pasteurized than in raw milk, with β-casein being slightly more susceptible to attack than αs1-easein. Measurements of γ-cascin and proteose-peptone formation have been made and it was found that PP5 was an intermediate product that was subject to further proteolysis while PP8F was a stable end-product. With the exception of component 3 (PP3), virtually all constituents of the proteose-peptone fraction increased during storage and appeared to be products of the action of proteolytic enzymes. Further evidence was obtained from the effects of various inhibitors that the principal proteinase of normal milk is plasmin, but slight differences were apparent between the protein breakdown patterns induced by storage and by added plasmin, which was consistent with the presence of more than one proteinase. Incubations in the presence of soya bean trypsin inhibitor to prevent plasmin action clearly revealed that another enzyme(s) was also involved.

Activation of latent collagenase from polymorphonuclear leukocytes by cathepsin G

1979 ◽  
Vol 44 (10) ◽  
pp. 3177-3182 ◽  
Author(s):  
Mária Stančíková ◽  
Karel Trnavský
Keyword(s):  
Affinity Chromatography ◽  
Trypsin Inhibitor ◽  
Polymorphonuclear Leukocytes ◽  
Soya Bean ◽  
Cathepsin G ◽  
Sepharose Column ◽  
Bean Trypsin Inhibitor ◽  
Human Polymorphonuclear Leukocytes

Cathepsin G was isolated from human polymorphonuclear leukocytes and purified by affinity chromatography on Antilysin-Sepharose column. Purified enzyme activated later collagenase isolated from leukocytes. Activation at 36°C was maximal after 30 min incubation. Inhibitors of cathepsin G - soya-bean trypsin inhibitor, diisopropyl phosphofluoridate and Antilysin were active in inhibiting the activation of latent collagenase by cathepsin G.

The formation and structure of some proteose-peptone components

1979 ◽  
Vol 46 (2) ◽  
pp. 215-218 ◽  
Author(s):  
Anthony T. Andrews
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Bovine Milk ◽  
Raw Milk ◽  
Terminal Part ◽  
Proteose Peptone ◽  
Proteolytic Cleavages

SUMMARYTwo constituents of the proteose-peptone fraction of bovine milk have been isolated and characterized. Component 5 (PP5) has been shown to represent residues 1–105 and 1–107 of the β-casein amino acid sequence, while component 8-fast (PP8F) corresponds to residues 1–28 of β-casein. Thus, these proteose-peptones represent the N-terminal portions of the β-casein molecule, produced by proteolytic cleavages which form the γ1-, γ2- and γ3-caseins from the C-terminal part. The continuing formation of the total proteose-peptone fraction, PP5, PP8F and the γ-caseins during storage of raw milk at 18 or 37 °C has also been demonstrated

scholarly journals Surface-simulation synthesis of the substrate-binding site of an enzyme. Demonstration with trypsin

1985 ◽  
Vol 226 (2) ◽  
pp. 477-485 ◽  
Author(s):  
M Z Atassi
Keyword(s):  
Binding Site ◽  
Trypsin Inhibitor ◽  
Binding Activity ◽  
Enzymic Activity ◽  
Soya Bean ◽  
Free Form ◽  
Protein Substrates ◽  
Substrate Analogues ◽  
Bean Trypsin Inhibitor ◽  
Alpha 1 Antitrypsin

From the X-ray co-ordinates of bovine trypsin and its complexes with substrate analogues (benzamidine) and with soya-bean trypsin inhibitor, a peptide (TP) was designed and synthesized by surface-simulation synthesis, a concept previously introduced by this laboratory, to mimic the binding site of trypsin. Also, a control peptide (CTP) was synthesized that contained all the amino acids present in the TP peptide, except that their order was randomized. The radioiodinated TP peptide bound specifically to adsorbents of benzamidine, whereas the control CTP peptide exhibited no binding activity. Conjugates to succinyl (3-carboxypropionyl)-lysozyme of the TP peptide, control CTP peptide and other unrelated peptides were examined by a radiometric binding assay for the ability to bind soya-bean trypsin inhibitor and human alpha 1-antitrypsin. Conjugates of the TP peptide exhibited considerable binding activity to adsorbents of soya-bean trypsin inhibitor or alpha 1-antitrypsin. None of the other peptide conjugates possessed any binding activity. Action of the active-site-directed reagents phenylmethanesulphonyl fluoride and di-isopropyl phosphorofluoridate on free TP and CTP peptides resulted in the modification of a serine residue in the TP peptide whereas the CTP peptide remained unaltered. The TP peptide, either in the free form or as a conjugate on succinyl-lysozyme, had no enzymic activity on protein substrates or on tosylarginine methyl ester. These findings indicated that the binding activity of an enzyme was well mimicked by the surface-stimulation peptide but that reproduction of the catalytic activity was not obtained.

An Endogenous Protease Activating Plasma Inactive Renin

1978 ◽  
Vol 55 (s4) ◽  
pp. 133s-134s ◽  
Author(s):  
B. J. Leckie
Keyword(s):  
Human Plasma ◽  
Serine Protease ◽  
Protease Inhibitors ◽  
Trypsin Inhibitor ◽  
Soya Bean ◽  
Acid Activation ◽  
Bean Trypsin Inhibitor ◽  
Inactive Renin ◽  
Inhibited Acid ◽  
Endogenous Protease

1. The protease inhibitors Trasylol and soya-bean trypsin inhibitor prevented the activation of plasma inactive renin by acid. 2. N-Ethylmaleimide inhibited acid-activation to some extent but o-phenanthroline had no effect. 3. Acid-activation of the inactive renin in human plasma is mediated by a serine protease.

scholarly journals Functional modifications of α2-macroglobulin by primary amines. Kinetics of inactivation of α2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin

1982 ◽  
Vol 201 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Fred Van Leuven ◽  
Jean-Jacques Cassiman ◽  
Herman Van Den Berghe
Keyword(s):  
Conformational Change ◽  
Trypsin Inhibitor ◽  
Binding Capacity ◽  
Soya Bean ◽  
Primary Amines ◽  
Slow Phase ◽  
Subunit Structure ◽  
Α2 Macroglobulin ◽  
Bean Trypsin Inhibitor ◽  
Kinetics Of

The unique steric inhibition of endopeptidases by human α2M (α2-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by α2M was closely correlated with the loss of the methylamine-reactive sites in α2M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/α2M ratios, no unaccounted loss of trypsin-binding capacity occurred. As α2M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form α2M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [14C]methylamine with α2M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of α2M with time of methylamine treatment. It was found that conformational change of α2M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of 125I-labelled trypsin to α2M did follow the same kinetics as the conformational change. This discrepancy between total binding (125I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound 125I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of α2M and to the mechanism of formation of the complex.

Peptidases from two strains ofPseudomonas fluorescens: partial purification, properties and action on milk

1987 ◽  
Vol 54 (2) ◽  
pp. 283-293 ◽  
Author(s):  
Kazi Shamsuzzaman ◽  
Robin C. McKellar
Keyword(s):  
Raw Milk ◽  
Subcellular Fractionation ◽  
Soya Bean ◽  
Partial Purification ◽  
Peptidase Activity ◽  
Ph Optimum ◽  
Bean Trypsin Inhibitor ◽  
Sterilized Milk ◽  
Low Activity ◽  
Sulphonyl Fluoride

SummaryPseudomonas fluorescensstrains 240 and 32A expressed cell-associated peptidase activity which was shown by subcellular fractionation to be primarily intracellular. Two peptidases were partly purified from strain 32A. One specifically hydrolysedN-α-benzoyl-DL-arginine-4-nitroanilide and was termed endopeptidase and the other hydrolysed L-lysine- and L-leucine-4-nitroanilide and was termed aminopeptidase. The endopeptidase had very low activity on bovine serum albumin compared with that of trypsin and probably was not a proteinase. The endopeptidase had a mol. wt of 33000 and a pH optimum of 8·0. The enzyme was stimulated by Ca2+and Mg2+and inhibited by Co2+, Mn2+, Hg2+, Zn2+and leupeptin. Soya bean trypsin inhibitor and phenylmethane sulphonyl fluoride (PMSF) had no effect on its activity. The aminopeptidase had a mol. wt of 44000 and a pH optimum of 8·0. It was inhibited by all the metal ions mentioned above and by PMSF. Little proteolysis was found when ultra high temperature (UHT) sterilized milk was treated with cell-free extract from strain 32A. It was concluded that the cell-associated peptidases fromPseudomonasstrains normally present in raw milk may not contribute significantly to the deterioration of UHT sterilized milk.

Measurement of Endotoxin Levels in Plasma Using Limulus Amoebocyte Lysate and Chromogenic Substrate, S2222

1979 ◽  
Author(s):  
S Clark ◽  
M Scully ◽  
P Webb ◽  
V Kakkar
Keyword(s):  
Trypsin Inhibitor ◽  
Final Concentration ◽  
Soya Bean ◽  
Lag Phase ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Linear Calibration ◽  
Limulus Amoebocyte Lysate ◽  
Bean Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor

A method has been devised for the measurement of endotoxin in plasma using the chromogenic substrate, S2222, a substrate which has been shown to be particularly sensitive to the Limulus Lysate. Time curves of the rate of release of the chromogen in mixtures in which procoagulase activation was concurrent, were complex with a lag phase which was shortened by increasing endotoxin concentrations. At a final concentration of 0.5ng/ml and 370 activation was complete within 60 minutes. The enzyme was inhibited by soya bean trypsin inhibitor but not by pancreatic trypsin inhibitor or hirudin. In the method finally adopted the lysate (25µl) was incubated with endotoxin (E.coli 026.86 Difco) and magnesium chloride (final concentration 33mM) in a total volume of 225µl. After 12 minutes preincubation 165µl of S2222(0.4mM) was added and the increase in abdorbance at 405nm over two minutes measured using an Abbott Biochromatic Analyser 100. Linear assay curves were obtained with final concentration of 0.2 to 2.0ngs endotoxin/ml with ΔOD 405/min of 0.35 at 2.0ng endotoxin/ml of final incubation mixture. ΔOD /min in control tubes were of the order of 0.02. For measurement from plasma samples, endotoxin was first extracted with chloroform. Linear calibration curves were achieved at a concentration of endotoxin of 1 to 5ng/ml of whole blood with a net OD/min at the highest concentration of 0.25.

Luminal Epidermal Growth Factor is Trophic to the Small Intestine of Parenterally Fed Rats

1995 ◽  
Vol 89 (2) ◽  
pp. 117-120 ◽  
Author(s):  
Tania Marchbank ◽  
Robert A. Goodlad ◽  
Chung Y. Lee ◽  
Raymond J. Playford
Keyword(s):  
Small Intestine ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Parenteral Nutrition ◽  
Trypsin Inhibitor ◽  
Soya Bean ◽  
Trophic Effect ◽  
Bean Trypsin Inhibitor ◽  
Epidermal Growth ◽  
Intestinal Atrophy

1. Intestinal atrophy contributes to the clinical difficulties of patients on parenteral nutrition. Systemic administration of epidermal growth factor reverses this effect, but there is concern over the clinical safety of intravenous administration of growth factors. We therefore investigated whether administration of luminal epidermal growth factor could reverse the atrophy induced in a rat model of parenteral nutrition when epidermal growth factor was given alone or in combination with soya bean trypsin inhibitor to reduce proteolytic digestion of the epidermal growth factor. 2. Infusion of soya bean trypsin inhibitor alone decreased intraluminal tryptic activity by about 90% but did not result in increased proliferation. Intragastric infusion of epidermal growth factor (72 μg/day per rat) caused a 26% increase in proliferation (determined by 2-h metaphase arrest) in the duodenum (P < 0.01) when compared with animals receiving ‘control’ intragastric infusion. However, intragastric epidermal growth factor had no effect on more distal regions of the bowel, probably reflecting rapid proteolysis of the epidermal growth factor by luminal proteases. In contrast, a trophic effect of luminal epidermal growth factor was seen in the duodenum (28% increase, P < 0.01) and jejunum (24% increase, P < 0.05) of animals which had received epidermal growth factor with soya bean trypsin inhibitor. This was probably due to the soya bean trypsin inhibitor decreasing the rate of degradation of epidermal growth factor by intestinal proteases, allowing biologically active epidermal growth factor to reach more distal portions of the bowel. 3. We conclude that luminal administration of epidermal growth factor in combination with protease inhibitors could provide a novel approach to the treatment of intestinal atrophy induced by parenteral nutrition. This approach may also be useful for the treatment of ulceration of the small intestine in conditions such as necrotizing enterocolitis or Crohn's disease. Clinical studies are recommended.

The roles of native milk proteinase and its zymogen during proteolysis in normal bovine milk

1982 ◽  
Vol 49 (4) ◽  
pp. 577-585 ◽  
Author(s):  
Olivier de Rham ◽  
Anthony T. Andrews
Keyword(s):  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bovine Milk ◽  
Raw Milk ◽  
Significant Activity ◽  
Peptide Bonds ◽  
Pasteurized Milk ◽  
Proteose Peptone ◽  
Heated Milk ◽  
Future Work

SUMMARYProteolysis was measured quantitatively in normal bulk milk, either raw, pasteurized or heated (95 °C, 15 min). During incubation at 37 °C for 24 h, about 0·7 mM of peptide bonds were split in raw milk, and 1·8 mM after activation of the zymogen with urokinase. The same values were observed in pasteurized milk, and no significant activity was present in heated milk. When compared with a commercial plasmin preparation, these levels correspond to about 1·4 and 3·6μg/ml of plasmin respectively. Most of this activity was separated in the micellar fraction, and it was suppressed by addition of soyabean trypsin inhibitor (SBTI). The remaining activity in the serum phase was not inhibited by SBTI and gave a rather non-specific breakdown with few well-defined casein fragments being produced. Upon further incubation, after the first 24 h, the activity increased, indicating that activation of the zymogen (plasminogen) occurred spontaneously. The rate of this activation was independent of the addition of more plasminogen and was higher in pasteurized than in raw milk. In pasteurized milk, all the native milk proteinase was in the form of the zymogen at the time of secretion. β-Casein was the preferred substrate for the milk proteinase (plasmin) and produced γ-caseins and proteose-peptone components 5 and 8-fast; other fragments were clearly visible on polyacrylamide gel electrophoresis, and included degradation products of αs1-casein. The formation of all these fragments was enhanced by addition of urokinase alone, or of plasminogen and urokinase, or by increasing the incubation time. They were also produced by incubating the micellar fraction alone, but not the serum fraction. Additional fragments were produced when porcine plasmin was added presumably due to differences in specificity between the porcine and bovine enzymes or to contaminating enzymes. Proteolysis induced by additions of plasminogen alone, or of plasminogen plus urokinase, was closer to that observed for the native milk proteinase, and must be recommended for future work in which it is desired to enhance the level of proteinase without altering breakdown patterns, unless a very pure bovine plasmin is available.

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