Willem Nieuwenhuizen

Purification and properties of phospholipase a from porcine pancreas

1. 1. Freshly prepared homogenates of pig pancreatic tissue contain a small amount of phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4); during autolysis, however, a considerable rise in lipolytic activity occurs. 2. 2. By use of heat treatment, (NH4)2SO4 precipitation and chromatographic procedures, the enzyme has been purified about 200 times and characterized by chemical and enzymatic procedures. 3. 3. The protein, which has a molecular weight of about 13 800 ± 500 appears to consist of one single polypeptide chain terminating in alanine (NH2) and cystine (COOH), and cross-linked intramolecularly by 7 disulphide bridges. 4. 4. The enzyme acts stereospecifically on all common types of 3-sn-phosphoglycerides, hydrolysing exclusively fatty acid ester bonds at the glycerol-C-2 position, regardless of chain length or degree of unsaturation. In contrast to the snake venom phospholipase A, the pancreatic enzyme shows a marked preference for anionic phospholipids such as phosphatidic acid, cardiolipin and phosphatidyl glycerol.

[15] Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from porcine pancreas

Publisher Summary Phospholipase A is present in high concentrations in the venoms of snakes and the bee. Several extensive purification procedures have been reported. In general the enzyme is characterized by a low molecular weight, a high content of disulfide bridges, and high heat stability. Most mammalian tissues have been shown to contain the enzyme; the highest concentration is found in the pancreas, where the enzyme is produced for digestive purposes. Like the proteolytic enzymes, phospholipase A is produced and secreted not in the active form but as enzymatically inactive zymogen. To demonstrate its presence in fresh pancreatic juice or in fresh pancreatic tissue, activation by limited tryptic hydrolysis is required. Up to now the most attractive source from which to isolate the enzyme seems to be the porcine pancreas. The starting material can easily be obtained in large amounts, and on the average one pig pancreas contains more than 50 mg of pure enzyme in the zymogen form.

Purification and properties of an anionic zymogen of phospholipase a from porcine pancreas

Abstract 1. 1. This paper describes the isolation and purification of an enzymically inactive precursor of porcine pancreatic phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4). 2. 2. The protein, which has a molecular weight of about 15 000, appears to consist of a single polypeptide chain, terminating at the NH 2 region in the amino acid sequence: Glu-Gly-Glu-Ile-Ser-Ser-Arg-Ala......, and having cystine as COOH-terminal amino acid. 3. 3. The precursor molecule is activated by trypsin which splits the above -Arg-Ala-peptide bond, yielding active phospholipase A and the heptapeptide: Glu-Gly-Glu-Ile-Ser-Ser-Arg. 4. 4. In this released peptide, as well as in the precursor molecule itself, the N-terminal glutamic acid residue has no free α-NH 2 group. 5. 5. Phospholipase A, isolated from autolysed pancreatic tissue, appears to be identical with the product obtained by trypsin activation of the pure precursor.

Plasminogen activation by tissue activator is accelerated in the presence of fibrin(ogen) cyanogen bromide fragment FCB-2

Fibrin, in contrast to fibrinogen, strongly accelerates the plasminogen activation by extrinsic activator (tissue-type plasminogen activator, t-PA). However, when fibrin and fibrinogen are digested with cyanogen bromide, both digests potentiate the t-PA-mediated plasminogen activation equally well. In this report, evidence is presented that this potentiating activity resides in CNBr fragment FCB-2 (= Ho1-DSK) and that a polymeric structure such as fibrin is not a prerequisite for the potentiation.

Identification of a site in fibrin(OGEN) which is involved in the acceleration of plasminogen activation by tissue-type plasminogen activator

The rate of activation of plasminogen by tissue-type plasminogen activator is greatly increased by fibrin, but not by fibrinogen. A possible explanation for this phenomenon could be that conformational changes take place during the transformation of fibrinogen to fibrin which lead to exposure of sites involved in the accelerated plasmin formation. This is also supported by our recent observation that some enzymatically prepared fragments of fibrinogen and fibrin (D EGTA, D-dimer, Y) and also CNBr fragment 2 from fibrinogen have this property. CNBr fragment 2 consists of amino acid residues A alpha (148-207), B beta (191-224) + (225-242) + (243-305) and gamma 95-265, kept together by disulphide bonds. In order to study the localization of a stimulating site within this structure we purified the chain remnants of CNBr fragment 2 after reduction and carboxymethylation, and found that only A alpha 148-207 was stimulating. This was further confirmed by digesting pure A alpha-chains with CNBr and purifying the resulting A alpha-chain fragments. CNBr digests of B beta- and gamma-chains were not stimulatory. The A alpha-chain remnant (residues 111-197) in D EGTA and D-dimer also comprise the major part (residues A alpha 148-197) of the CNBr A alpha-chain fragment. We conclude that a site capable of accelerating the plasminogen activation by tissue-type plasminogen activator preexists in fibrinogen, that this site becomes exposed upon fibrin formation or disruption of fibrinogen by plasmin or CNBr and that this site is within the stretch A alpha 148-197, which is retained in the A alpha-chain remnants of fibrinogen degradation products.

Direct evidence of transcriptional control of fibrinogen and albumin synthesis in rat liver during the acute phase response

Summary Using immunoprecipitation technique we have purified fibrinogen polypeptide mRNAs to homogeneity as demonstrated by translation in a wheat germ cell free system and by hybridization kinetics. By measuring the sequence contents of fibrinogen polypeptide mRNAs and albumin mRNA, using tritiated DNAs complementary to fibrinogen polypeptide mRNAs and to albumin mRNA respectively, a dramatical increase in mRNA content of fibrinogen polypeptides (7 fold) and a decrease in albumin mRNA content (2 fold) have been found in the liver of rats 24 hours after i.m. injection of 1.0 ml turpentine. These results were consistent with the findings in cell free translation under the direction of poly A + RNA prepared from livers of experimental animals and suggest that the fibrinogen and albumin synthesis during the acute phase response is reciprocally regulated at the transcriptional level.

Fibrin-Mediated Plasminogen Activation

Abstract: Fibrin, but not fibrinogen, enhances the rate of activation of plasminogen by tissue type plasminogen activator (t‐PA). Studies with enzymatic and chemical fragments of fibrinogen showed that several sites in fibrinogen are involved in this rate enhancement; these are, Aα148–160 (located in CNBr fragment FCB‐2), and FCB‐5 (a CNBr fragment comprising γ312–324), and recently discovered sites in the fibrinogen αC domains. All these sites are buried in fibrinogen, but exposed in fibrin and some fibrinogen fragments. For the first two of these, located in the D‐domains, this was shown by the fact that monoclonal antibodies against Aα148–160 and γ312–324 bind to fibrin and rate enhancing fibrin(ogen) fragments, but not to fibrinogen. Direct binding studies indicate that at physiological concentrations plasminogen binds to FCB‐2, and t‐PA to FCB‐5. More detailed studies have demonstrated the importance of residues Aα‐157 and Aα‐152, and that the minimum stretch with rate enhancing properties is Aα154–159. The sites in the αC domains await further identification. With the recently reported three‐dimensional structure of fragments D and D‐dimer it is now possible to explain these findings at the molecular level. Molecular calculations and experimental data show that the site Aα148–160 in fibrinogen is covered among others by a part of the Aα chain (Aα166–195) that forms an α‐helix, and by a globular domain formed by the β‐chain. On fibrin formation, the last two may move away, and give access to Aα148–160. It is conceivable that in the αC domain sites are involved in the early phases of fibrinolysis. The site Aα148–160 and that in FCB‐5 may be more important at later stages. It is also clear that fibrin polymerization is important. This polymerization has probably several effects: exposure of the rate enhancing sites; mutual positioning of the t‐PA and plasminogen binding sites; a concentrating effect of t‐PA and plasminogen on the fibrin surface; effects on the kinetic properties of t‐PA and plasminogen. These effects together explain the rate enhancement.

Studies on phospholipase A and its zymogen from porcine pancreas: II. The assignment of the position of the six disulfide bridges

Abstract Porcine pancreatic phospholipase A and its zymogen are single-chain proteins consisting of 123 and 130 amino acids, respectively. Both proteins contain twelve half-cystine residues and the absence of free sulfhydryl groups indicates the presence of six disulfide bridges. To assign the positions of these cystine bonds in the protein, enzymatic digestion was performed with pepsin, chymotrypsin and thermolysin under conditions which minimize disulfide interchange. After isolation of the cystine-containing peptides, the positions of the six disulfide bonds were established.

Evidence of Fibrinogen Breakdown by Leukocyte Enzymes in a Patient with Acute Promyelocytic Leukemia

On daunomycin treatment of a patient with promyelocytic leukemia, leukocyte elastase appeared in large amounts in the patients blood. Also, the plasma fibrinogen was found to be partially degraded to early, X-like, fibrinogen degradation products. These early fibrinogen fragments were isolated and showed a low anticoagulant activity in a thrombin time test. Early fibrinogen degradation products, produced with leukocyte elastase in vitro, have a similar low anticoagulant activity. In contrast, plasmic degradation products inhibit clotting of fibrinogen to a large extent. Although alpha 2-antiplasmin and plasminogen levels were low, antithrombin III levels were not decreased. The low anticoagulant activity of the isolated fibrinogen fragments, the presence of elastase activity in the plasma--both immunological and amidolytic--and the normal levels of antithrombin III suggest that granulocytic enzymes, whose release was enhanced by the cytostatic treatment, were responsible for degradation of fibrinogen in this patient.

Calcium-binding properties of human fibrin(ogen) and degradation products.

Here the results obtained with human fibrinogen and its degradation products are reported. Our results differ from those obtained in (9) but strongly support model suggested earlier by us for Ca2(+)-binding by rat fibrinogen. Chemicals/CAS: calcium, 7440-70-2; fibrin, 9001-31-4; fibrinogen, 9001-32-5; Calcium, 7440-70-2; Fibrin, 9001-31-4; Fibrinogen, 9001-32-5; Macromolecular SystemsChemicals/CAS: calcium, 7440-70-2; fibrin, 9001-31-4; fibrinogen, 9001-32-5; Calcium, 7440-70-2; Fibrin, 9001-31-4; Fibrinogen, 9001-32-5; Macromolecular Systems