Caseinolytic Units Research Articles

Peptidases from Maclura Pomifera for Preparation of Food Protein Hydrolysates: Purification by Single-Step Chromatography and Characterization of Pomiferin I.

Our objective was to isolate peptidases from the latex of Maclura pomifera fruits and use them to hydrolyze food proteins, as well as to purify and characterize the main peptidase. Two partially purified proteolytic extracts were prepared by ethanol (EE) and acetone (AE) precipitation from an aqueous suspension of exuded fruit latex. EE was used to hydrolyze food proteins with a ratio of 0.19 caseinolytic units (Ucas) per mg of substrate. Different values of hydrolysis degree were observed for hydrolysates of egg white, soy protein isolate, and casein at 180min (9.3%, 31.1%, and 29.1%, respectively). AE was employed to purify a peptidase which exhibited an isoelectric point (pI) of 8.70 and whose abundance in AE was 28.3%. This enzyme was purified to homogeneity using a single-step procedure by cation-exchange chromatography, achieving an 8.1-fold purification and a yield of 16.7%. The peptidase was named pomiferin I and showed a molecular mass of 63,177.77Da. Kinetic constants (KM 0.84mM, Vmax 27.50 uM s-1, kcat 72.37s-1, and kcat/KM 86.15mM-1s-1) were determined employing N-α-carbobenzoxy-L-alanyl-p-nitrophenyl ester as substrate. Analysis by PMF showed only partial homology of pomiferin I with a serine peptidase from a species of the same family.

Blood metabolites, meat quality and muscle calpain–calpastatin activities in goats treated with low doses of recombinant bovine somatotropin

Growing female Alpine goats ( n = 14; 6 month of age; BW of 18 ± 2.7 kg) were used in a 10-week experiment to assess the effect of daily injection of a low dose (40 μg/kg of BW) of recombinant bovine somatotropin (rbST) on blood metabolite concentrations, mass of the visceral organs, the calpain system, and chevon quality. Goats were randomly assigned to either a control (0.9% saline; n = 7) or a treatment group ( n = 7) receiving a daily dose of rbST. All animals were fed a 16% CP and 2.9 Mcal/kg DE alfalfa based-diet ad libitum. Blood samples were collected every 2 weeks for a 10-week period and analyzed for glucose, NEFA, and PUN. At the end of the 10-week period, animals were sacrificed and processed. A portion of the loin muscle was removed, trimmed of fat, denuded and homogenized in an extraction buffer for determination of the calpain–calpastatin system. Components of the viscera were separated and individually weighed. The carcass was chilled for 24 h at 2 °C. Before cooking the loin/rib chops, instrumental color measurements were done after 45 min bloom. Shear values were determined on cooked chops. Plasma non-esterified fatty acid concentrations were influenced by treatment ( P < 0.01), sampling time ( P < 0.01), and treatment × sampling time interaction ( P < 0.01). Plasma glucose and PUN concentrations increased over sampling time ( P < 0.01). At the end of the 10-week treatment period, PUN was higher in goats treated with rbST than in controls. Body weight change, carcass weight, visceral mass, and loin eye area were not influenced by rbST treatment. Mean m-calpain, μ-calpain, and calpastatin activities (caseinolytic units per 50 g of tissue), as well as pre-aging instrumental color ( L *, a *, b *, chroma and Hue) values were also not affected by rbST injections. While treatment or treatment × aging time interaction did not influence cooking loss, aging decreased ( P < 0.01) shear value but without significant improvement beyond 7 days. There was a positive correlation between longissimus muscle final pH and m-calpain activities in both bST-treated and control goats ( P < 0.05), although the relationship between final pH and calpastatin activities was much stronger ( P < 0.02). Daily injection of a low dose of rbST did not alter the meat quality, muscle calpain system, or weights of visceral organs.

Contact Activation Triggers Stimulation of the Monocyte 5-Lipoxygenase Pathway Via Plasmin

AbstractThe purpose of this study was to characterize the stimulus that activates the 5-lipoxygenase pathway in human peripheral monocytes (PM) during the process of contact activation. Incubation of PM, but not of polymorphonuclear leukocytes (PMN), in contact-activated, recalcified plasma induced a time-dependent release of leukotrienes (LT). The presence of platelets was required for the generation of cysteinyl-LT, but LTB4 formation also proceeded in their absence, although to a lesser extent. Plasmin, presumably generated via the intrinsic fibrinolytic pathway, was liable for the 5-lipoxygenase stimulation during contact activation inasmuch as (1) the 5-lipoxygenase pathway in PM was stimulated by contact-activated, recalcified, autologous or homologous plasma, but not by factor XII-deficient or prekallikrein-deficient plasma; (2) lysine analogs such as Nα-acetyl-L-lysine, 6-aminohexanoic acid (6-AHA), or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), which inhibit plasmin(ogen) binding to PM plasmin(ogen) binding sites, concentration-dependently reduced the cyst-einyl-LT release; (3) plasminogen activators such as urokinase or streptokinase concentration-dependently enhanced the cysteinyl-LT release up to 10 and 1,000 lU/mL, respectively, while higher concentrations were less effective leading to bell-shaped concentration-response curves; (4) plasmin inhibitors such as aprotinin or α2-antiplasmin concentration-dependently inhibited the cysteinyl-LT release; and (5) preincubation of plasma with monoclonal antibodies directed against plasminogen and capable of preventing plasminogen activation blocked the contact-mediated 5-lipoxygenase stimulation. Moreover, incubation of PM with plasmin, but not with plasma kallikrein, in Hanks’ balanced salt solution (HBSS)-bovine serum albumin (BSA) 0.4% triggered a concentration-dependent release of LTB4 up to 0.1 caseinolytic units (CU)/mL, with higher concentrations being less effective. By contrast, release of cyclooxygenase metabolites such as thromboxane (TX) B2 and prostaglandin (PG) E2 was not stimulated by plasmin, indicating specificity for the 5-lipoxygenase pathway. With plasmin as a hitherto unknown stimulus of the 5-lipoxygenase pathway in PM, a novel link between contact activation and inflammation has been established.

Contact activation triggers stimulation of the monocyte 5-lipoxygenase pathway via plasmin

The purpose of this study was to characterize the stimulus that activates the 5-lipoxygenase pathway in human peripheral monocytes (PM) during the process of contact activation. Incubation of PM, but not of polymorphonuclear leukocytes (PMN), in contact-activated, recalcified plasma induced a time-dependent release of leukotrienes (LT). The presence of platelets was required for the generation of cysteinyl-LT, but LTB4 formation also proceeded in their absence, although to a lesser extent. Plasmin, presumably generated via the intrinsic fibrinolytic pathway, was liable for the 5-lipoxygenase stimulation during contact activation inasmuch as (1) the 5-lipoxygenase pathway in PM was stimulated by contact-activated, recalcified, autologous or homologous plasma, but not by factor XII-deficient or prekallikrein- deficient plasma; (2) lysine analogs such as N alpha-acetyl-L-lysine, 6- aminohexanoic acid (6-AHA), or trans-4- (aminomethyl)cyclohexane-1- carboxylic acid (t-AMCA), which inhibit plasmin(ogen) binding to PM plasmin(ogen) binding sites, concentration-dependently reduced the cysteinyl-LT release; (3) plasminogen activators such as urokinase or streptokinase concentration-dependently enhanced the cysteinyl-LT release up to 10 and 1,000 IU/mL, respectively, while higher concentrations were less effective leading to bell-shaped concentration- response curves; (4) plasmin inhibitors such as aprotinin or alpha 2- antiplasmin concentration-dependently inhibited the cysteinyl-LT release; and (5) preincubation of plasma with monoclonal antibodies directed against plasminogen and capable of preventing plasminogen activation blocked the contact-mediated 5-lipoxygenase stimulation. Moreover, incubation of PM with plasmin, but not with plasma kallikrein, in Hanks' balanced salt solution (HBSS)-bovine serum albumin (BSA) 0.4% triggered a concentration-dependent release of LTB4 up to 0.1 caseinolytic units (CU)/mL, with higher concentrations being less effective. By contrast, release of cyclooxygenase metabolites such as thromboxane (TX) B2 and prostaglandin (PG) E2 was not stimulated by plasmin, indicating specificity for the 5-lipoxygenase pathway. With plasmin as a hitherto unknown stimulus of the 5-lipoxygenase pathway in PM, a novel link between contact activation and inflammation has been established.

Temperature Dependence of Plasmin-Induced Activation or Inhibition of Human Platelets

It is known that at 37 degrees C plasmin may have two opposite effects on platelets: at high concentrations (greater than 1.5 caseinolytic units [CU]/mL), plasmin activates platelets; at lower concentrations (0.1 to 1.0 CU/mL) it inhibits platelet activation induced by thrombin, collagen, or calcium ionophore A23187. In this study, we report that when lowering the incubation temperature to 22 degrees C, plasmin at low concentrations (0.1 to 0.5 CU/mL) fully activated platelets. When platelets were treated with 0.2 CU/mL of plasmin, lowering the incubation temperature from 37 degrees C to 22 degrees C resulted in an increase in the expression of fibrinogen receptors, in platelet release and aggregation. Thromboxane A2 was not generated by plasmin treatment at either temperature. Ultrastructural studies showed that platelets responded to low-dose plasmin at 37 degrees C by forming pseudopods, centralizing granules without fibrinogen release, whereas at 22 degrees C the same dose of plasmin caused platelet degranulation with the appearance of alpha-granule fibrinogen within the lumen of the surface connected canalicular system. In addition, at 22 degrees C plasmin at doses insufficient to induce platelet aggregation potentiated platelet response to thrombin. Thus, we suggest that plasmin may initiate both activating and inhibitory processes within platelets and that the change of temperature could influence this balance. These results may be of clinical relevance, because the fibrinolytic system was found activated during cardiopulmonary bypass in which the temperature of patient's blood circulation was reduced. This temperature-dependent behavior is also an interesting model for a further study on platelet response to serine proteinases.

Temperature dependence of plasmin-induced activation or inhibition of human platelets

It is known that at 37 degrees C plasmin may have two opposite effects on platelets: at high concentrations (greater than 1.5 caseinolytic units [CU]/mL), plasmin activates platelets; at lower concentrations (0.1 to 1.0 CU/mL) it inhibits platelet activation induced by thrombin, collagen, or calcium ionophore A23187. In this study, we report that when lowering the incubation temperature to 22 degrees C, plasmin at low concentrations (0.1 to 0.5 CU/mL) fully activated platelets. When platelets were treated with 0.2 CU/mL of plasmin, lowering the incubation temperature from 37 degrees C to 22 degrees C resulted in an increase in the expression of fibrinogen receptors, in platelet release and aggregation. Thromboxane A2 was not generated by plasmin treatment at either temperature. Ultrastructural studies showed that platelets responded to low-dose plasmin at 37 degrees C by forming pseudopods, centralizing granules without fibrinogen release, whereas at 22 degrees C the same dose of plasmin caused platelet degranulation with the appearance of alpha-granule fibrinogen within the lumen of the surface connected canalicular system. In addition, at 22 degrees C plasmin at doses insufficient to induce platelet aggregation potentiated platelet response to thrombin. Thus, we suggest that plasmin may initiate both activating and inhibitory processes within platelets and that the change of temperature could influence this balance. These results may be of clinical relevance, because the fibrinolytic system was found activated during cardiopulmonary bypass in which the temperature of patient's blood circulation was reduced. This temperature-dependent behavior is also an interesting model for a further study on platelet response to serine proteinases.

Platelet protein phosphorylation, elevation of cytosolic calcium, and inositol phospholipid breakdown in platelet activation induced by plasmin.

Studies have been performed on the biochemical mechanism of platelet activation induced by the fibrinolytic protease plasmin. In washed human platelets, greater than or equal to 1.0 caseinolytic units (CU/ml plasmin induced aggregation. Platelet [14C]serotonin release was stimulated by 1.0 CU/ml plasmin to an extent comparable to that induced by 1.0 U/ml thrombin. A dose- and time-dependent phosphorylation of the platelet 47,000- and 20,000-kD proteins was noted in 32PO4-labeled platelets incubated with plasmin; phosphorylation was not affected by extracellular Ca2+, but was completely inhibited by an increase in platelet cyclic AMP. Phosphorylation of these platelet proteins suggested that plasmin may act on platelets by stimulating a rise in cytosolic calcium concentration ([Cai2+]) and activating inositol phospholipid-dependent phospholipase C and protein kinase C. Using both quin2 fluorescence and aequorin luminescence as indicators, plasmin was found to elevate platelet [Cai2+] in the presence or absence of extracellular Ca2+. Phospholipase C activation was shown by the generation of [3H]diglyceride in [3H]arachidonic acid-labeled platelets and [32P]phosphatidic acid in 32PO4 labeled platelets exposed to plasmin. Plasmin did not induce formation of thromboxane A2 (TXA2). Only small amounts of this eicosanoid were detected late in the time course after plasmin stimulation. Our results indicate that plasmin causes platelet aggregation and secretion associated with phosphorylation of the 47,000- and 20,000-kD proteins, Ca2+ mobilization, and phospholipase C and protein kinase C activation.

Studies on the role of polymorphonuclear leukocyte granule elastase in arthus reaction

In the present study, attempts were made to clarify the role of an elastase-like enzyme in Arthus reaction by using rabbit-peritoneal polymorphonuclear leukocytes (PMNs). It was first confirmed that the injection of large amounts of PMNs with antibody can increase tissue damage in reversed Arthus reaction. The five various fractions, which were intact PMNs, whole homogenate, granule fraction, nuclei and cell debris, and membrane extract, were prepared from rabbit-peritoneal exudate. Each fraction was intracutaneously injected into the abdominal wall of rabbit. It was of particular interest that the tissue damage induced by membrane extract occurred earlier and was more severe than that caused by the others. Furthermore, membrane extract possessed remarkably higher caseinolytic and elastinolytic activities than the others. The membrane extract was partially purified by Sephadex G-100 column chromatography. The protein concentration of the elastase-like enzyme solution was 3.64 mg/ml, and its specific activity was 195 caseinolytic units and 0.45 elastinolytic units per milligram of protein. The cutaneous reaction by membrane extract was dependent on the concentration of this elastase-like enzyme and could be strongly inhibited by Elastatinal. From these results, the authors suggest that an elastase-like enzyme which originates in PMN granules is the key enzyme that promotes the severity of tissue damage in Arthus reaction.

Plasminogen: purification from human plasma by affinity chromatography.

Plasminogen was prepared from human plasma by affinity chromatography on L-lysine-substituted Sepharose. Thirty milligrams of plasminogen, with a specific activity of 100 caseinolytic units (Committee on Thrombolytic Agents) per milligram of nitrogen, were obtained from 340 milliliters of plasma. This corresponds to over 200-fold purification from plasma. Disc-gel electrophoresis at pH 8.3 indicated seven distinct bands, all of which contained activity.

Determination of Plasminogen in Human Plasma by a Casein Method*

SummaryA modification of the casein method for determination of plasminogen in plasma has been devised.1. Urokinase instead of streptokinase was used for activation of plasminogen in order to eliminate the effect of any antistreptokinase antibodies in the plasma.2. A purified and stable plasmin preparation according to Wallén was used for preparing reference curves.3. The plasminogen activity of plasma was converted into arbitrary casein - olytic units (ACU) defined in such a way that 10 caseinolytic units gave an extinction of 0.300.4. Removal of the plasmin inhibitors by acidification of plasma to pH 2.0 was found to be better than using euglobulin precipitation.5. The plasma should be tested immediately after thawing and not allowed to stand after acidification for obtaining optimal conditions.6. The method can be used for determination of plasminogen in plasma in patients treated with ssss-ACA and AMCA.7. The error of the method was found to be small ssss0.4.The mean plasminogen level in a normal group of 72 was found to be 9.5 ssss 1.7 ACU/ml plasma. No variation with sex and age was found.The method proved clinically useful. During the latter half of pregnancy the plasminogen level was found to be clearly elevated. In patients with liver cirrhosis the plasminogen level was low. It was a sensitive method for following the plasminogen content during streptokinase treatment.

THE PLASMINOGEN-PLASMIN CONTENT OF UMBILICAL CORD BLOOD.

Summary:The plasminogen contents of the blood of the umbilical vessels and of the maternal peripheral vein were determined. The plasminogen content of the umbilical artery blood varied from 0.2 to 10.1 caseinolytic units per ml. of serum and that of the umbilical vein blood from 2.7 to 9.8 caseinolytic units per ml. of serum. In the maternal peripheral vein, the plasminogen content varied from 7.5 to 15.6 caseinolytic units per ml. of serum.The average fibrinogen content in the umbilical vein was 298 mg. per 100 ml. of plasma and in maternal peripheral plasma it was 487 mg. per 100 ml.In this series, where greater spontaneous activity was detected in the umbilical artery than in the umbilical vein, the plasminogen content of the umbilical artery was correspondingly lower than that of the umbilical vein. It is postulated that this is because of increased utilization of plasminogen.Since in many cases there was no difference in spontaneous ‐plasmin activity and plasminogen content in arterial and venous cord bloods, it is considered also that in the cases where there was increased plasmin activity and decreased plasminogen content a recent intervillous thrombosis had occurred.

THE EVALUATION IN THE DOG OF A PROTEOLYTIC ENZYME DERIVED FROM ASPERGILLUS ORYZAE.

A proteolytic enzyme (Aspergillin-O) derived from Aspergillus Oryzae was studied. For this purpose arterial and venous occluding thrombi in dogs were subjected to systemic treatment with the enzyme, and the value of Aspergillin-O in thrombolytic therapy, as well as its toxic side effects and some aspects of its pharmacology are described. Lysis of occluding arterial thrombi was achieved within a few hours after the administration of enzyme preparations by single intravenous injection, and arteries remained patent during the week of post-treatment observation. The thrombolytic dose was found to vary from animal to animal, but was generally comparable in its effect to a dose of human fibrinolysin of between 250 and 300 Michigan Department of Health caseinolytic units. The variations in dose requirements and toxicity were attributed to individual protease resistance, and a dose prediction test based on this concept is described. The dose prediction test was found to make possible the safe and efficient use of the enzyme in systemic thrombolytic therapy.

The Plasmin Inhibitors of Plasma

SummaryA new method for the assay of plasmin inhibitors in human plasma is described. The method consists of determination of the caseinolytic activity of a standard plasmin solution before and after incubation with the inhibitor, with lysine added to the mixture as a stabilizer of plasmin. Using this method, it was found that plasma contains enough inhibitors to inactivate 30 caseinolytic units of plasmin, or 10 times the normal amount of plasminogen in human plasma.

Colmn chromatography of human plasminogen on diethylaminoethyl cellulose.

Abstract The chromatography of acid-extracted samples of human plasminogen on DEAE-cellulose columns at 0°–4°, employing step-wise elution schemes, shows the grossly heterogeneous nature of such preparations. Quantitative analysis shows complete recovery of the total protein, but only 60–80% of the caseinolytic units applied to the column are recovered. The chromatography has been done in ammonium acetate buffers, containing 0.01 M l -lysine; such conditions have give rise to 6 peaks in the effluent pattern. Proteolytic assays indicate that approx. 40% of the original preparation is plasminogen. A dramatic change in the chromatogram took place when the concentration of l -lysine was raised to 0.05 M. The specific activity was increased by 5–10 times in one fraction. The chromatography has also been done with 6 M urea. These conditions have given 3 peaks in the resulting chromatogram, and complete recovery with respect to the total protein, but there was a partial loss of caseinolytic activity.

Column chromatography of human plasminogen on diethylaminoethyl cellulose

Abstract The chromatography of acid-extracted samples of human plasminogen on DEAE-cellulose columns at 0°–4°, employing step-wise elution schemes, shows the grossly heterogeneous nature of such preparations. Quantitative analysis shows complete recovery of the total protein, but only 60–80% of the caseinolytic units applied to the column are recovered. The chromatography has been done in ammonium acetate buffers, containing 0.01 M l -lysine; such conditions have give rise to 6 peaks in the effluent pattern. Proteolytic assays indicate that approx. 40% of the original preparation is plasminogen. A dramatic change in the chromatogram took place when the concentration of l -lysine was raised to 0.05 M. The specific activity was increased by 5–10 times in one fraction. The chromatography has also been done with 6 M urea. These conditions have given 3 peaks in the resulting chromatogram, and complete recovery with respect to the total protein, but there was a partial loss of caseinolytic activity.

HUMAN FIBRINOLYSIN WITH AND WITHOUT UROKINASE: ITS EFFECT ON EXPERIMENTAL ARTERIAL THROMBI IN DOGS

The lytic effects of heat-treated, sterile solutions of spontaneously activated and urokinase-activated fibrinolysin on occlusive experimental arterial clots have been studied. For this purpose methods for the preparation of urokinase and activation of profibrinolysin, the production of traumatic arterial thrombi in dogs, and the recording of clot lysis are described. Single injection as a means of administration was compared with the traditional method of infusion. When used in conjunction with a prompt assay of the in vivo fibrinolytic activity, which was developed for this purpose, the injection method was preferred. The results indicated that spontaneous fibrinolysin, given by single injection, was as effective in lysing clots as were preparations of fibrinolysin that contained activator. The approximate therapeutic dose for both types of fibrinolysin under the conditions described was found to be 225–325 Michigan Department of Health caseinolytic units per kg body weight. Lysis of occluding arterial thrombi was achieved within a few hours after administration of fibrinolysin by injection, and arteries remained patent during the week of posttreatment observation. No toxic effects due to the fibrinolysin or the glycerol present as stabilizer could be demonstrated after the therapeutic dose. The only undesirable side effect of overdose of fibrinolysin was a transient incoagulability of the blood.

Fibrinolysis. VIII. Comparative study in vitro of the effects of various proteolytic agents on the blood clotting mechanism of human plasma.

Abstract Nine enzymatic agents of different source and of different degree of purification were investigated with reference to their effect on the clotting mechanism and for their esterase activity in vitro. Comparable concentrations were used on the basis of the caseinolytic activity of each agent. The effects of the various materials were related, as expected, to the dose used. At a dose of 20 × 10 −3 caseinolytic units per milliliter all agents shortened the partial thromboplastin time. Some preparations (trypsin and protease, a bacterial agent) accelerated also the generation of thromboplastin and of thrombin. On the other hand, Aspergillin O, PK (a bacterial endopeptidase), and rhozyme A-4 (another fungal product from Aspergillus oryzae and niger) delayed the generation of thrombin. Aspergillin O and PK exhibited antithrombin III activity, while rhozyme A-4 delayed the plasma prothrombin time, presumably through the release of antithrombin VI activity. The effect of all enzymes of individual clotting agents was negligible unless high concentrations were used. On the basis of these studies, PK, Aspergillin O, and, to a lesser extent, rhozyme A-4 offered, promise as fibrinolytic agents because of their clot delaying effect associated with high fibrinolytic activity.

HUMAN FIBRINOLYSIN WITH AND WITHOUT UROKINASE: ITS EFFECT ON EXPERIMENTAL ARTERIAL THROMBI IN DOGS

The lytic effects of heat-treated, sterile solutions of spontaneously activated and urokinase-activated fibrinolysin on occlusive experimental arterial clots have been studied. For this purpose methods for the preparation of urokinase and activation of profibrinolysin, the production of traumatic arterial thrombi in dogs, and the recording of clot lysis are described. Single injection as a means of administration was compared with the traditional method of infusion. When used in conjunction with a prompt assay of the in vivo fibrinolytic activity, which was developed for this purpose, the injection method was preferred. The results indicated that spontaneous fibrinolysin, given by single injection, was as effective in lysing clots as were preparations of fibrinolysin that contained activator. The approximate therapeutic dose for both types of fibrinolysin under the conditions described was found to be 225–325 Michigan Department of Health caseinolytic units per kg body weight. Lysis of occluding arterial thrombi was achieved within a few hours after administration of fibrinolysin by injection, and arteries remained patent during the week of posttreatment observation. No toxic effects due to the fibrinolysin or the glycerol present as stabilizer could be demonstrated after the therapeutic dose. The only undesirable side effect of overdose of fibrinolysin was a transient incoagulability of the blood.