In vitro effect of elastase and cathepsin G from human neutrophils on creatine kinase and lactate dehydrogenase isoenzymes

1993 ◽  
Vol 39 (6) ◽  
pp. 986-992
Author(s):  
J A Stark ◽  
A R Henderson
Keyword(s):  
Creatine Kinase ◽  
Lactate Dehydrogenase ◽  
Hypochlorous Acid ◽  
Proteolytic Enzymes ◽  
Sodium Dodecyl ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Sample Treatment ◽  
Vitro Effect

Abstract The polymorphonuclear granulocyte, or neutrophil, has been implicated as a mediator of tissue-destructive events because it releases the preformed proteolytic enzymes elastase and cathepsin G, and, as a result of myeloperoxidase action, hypochlorous acid. We show that elastase inactivates and fragments creatine kinase isoenzymes CK-2 and CK-3, and, to a lesser extent, lactate dehydrogenase (LD) isoenzyme LD-1, whereas cathepsin G acts only on CK-2. Both neutrophil enzymes act on LD-3. The course of inactivation was followed by measuring the loss of catalytic activity at 37 degrees C. The evidence for fragmentation was obtained by gel filtration; electrophoresis after sample treatment with sodium dodecyl sulfate and 2-mercaptoethanol was less satisfactory for this purpose. Hypochlorous acid inactivates CK activity by about 75% at concentrations as low as 8 mumol/L and totally at concentrations > 140 mumol/L, whereas LD activity is not affected until concentrations exceed 200 mumol/L. After a myocardial infarction, the number of neutrophils increases; they are triggered and concentrate around damaged myocardial tissue. Our data suggest that neutrophils may inactivate and fragment "cardiac" enzymes released from such damaged tissue.

Interleukin-8 induces neutrophil accumulation but not protease secretion in the canine trachea

1992 ◽  
Vol 263 (6) ◽  
pp. L708-L713 ◽  
Author(s):  
P. G. Jorens ◽  
J. B. Richman-Eisenstat ◽  
B. P. Housset ◽  
P. D. Graf ◽  
I. F. Ueki ◽  
...  
Keyword(s):  
Interleukin 8 ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Secretory Cells ◽  
Neutrophil Accumulation ◽  
High Concentrations ◽  
Granule Secretion ◽  
Protease Secretion

The neutrophil enzyme elastase is a potent secretagogue of airway secretory cells, and elastase is present in high concentrations in sputum of patients with hypersecretion (e.g., cystic fibrosis, bronchiectasis). Interleukin-8 (IL-8), a recently discovered cytokine with potent neutrophil chemotactic properties in vitro, is also found in the sputum of these patients. We used an isolated tracheal segment in dogs in vivo to study the effect of IL-8 in causing neutrophil accumulation, elastase release, and secretion (by measuring lysozyme concentrations) in the luminal superfusate. IL-8 caused a potent time-dependent neutrophil accumulation at between 3 and 6 h. The effect was significant at 10(-9) and maximum at 10(-8) M. No increase in free elastase, cathepsin G, or lysozyme was detected in the superfusate. Thus, in contrast to previous studies showing that ragweed antigen causes the accumulation of neutrophil elastase which in turn causes lysozyme secretion, IL-8 causes neutrophil accumulation without granule secretion (or subsequent secretagogue activity). The findings were confirmed with dog and human neutrophils in vitro.

Iohexol-Induced Neutrophil Myeloperoxidase Release and Activation upon Contact with Vascular Stent-Graft Material: A Mechanism Contributing to the Postimplantation Syndrome?

2003 ◽  
Vol 10 (5) ◽  
pp. 958-967 ◽  
Author(s):  
Vibeke Videm ◽  
Asbjørn Ødegård ◽  
Hans Olav Myhre
Keyword(s):  
Stent Graft ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Maximal Response ◽  
Graft Material ◽  
Vascular Stent ◽  
Neutrophil Degranulation ◽  
Considerable Individual Variation ◽  
Vitro Effect

Purpose: To investigate whether the contrast medium iohexol alone or in combination with vascular stent-graft material induces neutrophil degranulation. Methods: Human whole blood or isolated human neutrophils were incubated with or without iohexol and vascular stent-graft material. Samples were also drawn from 5 patients undergoing diagnostic angiography using iohexol. Myeloperoxidase and lactoferrin concentrations were determined by enzyme immunoassays. Results: Both in vitro and in the patients, iohexol induced neutrophil degranulation with considerable individual variation in dose sensitivity and timing. The in vitro effect was independent of the type of anticoagulant used (ethylenediamine tetra-acetic acid, heparin, lepirudin). Experiments using isolated neutrophils showed that degranulation was independent of complement activation or platelet-derived mediators. The dose for maximal response varied from 5 to 50 mg I/mL (10.7–107.6 mg/mL iohexol). In vitro, vascular stent-graft material alone did not induce neutrophil degranulation. As compared to iohexol alone, incubation with iohexol and vascular stent-graft material in combination substantially increased the release of myeloperoxidase. Conclusions: Iohexol induces neutrophil degranulation, which is greatly enhanced when combined with vascular stent-graft material. Thus, iohexol-induced neutrophil activation may contribute to an inflammatory response following stent-graft implantation. We speculate that neutrophil activation during other procedures combining catheters and iohexol (e.g., angiography) may induce inflammation, which might have detrimental effects.

Effects of Some Vitamins on Lactoperoxidase Enzyme Activity

2009 ◽  
Vol 79 (3) ◽  
pp. 188-194 ◽  
Author(s):  
Melda Sisecioglu ◽  
Murat Cankaya ◽  
Hasan Ozdemir
Keyword(s):  
Ascorbic Acid ◽  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Exchange Chromatography ◽  
Inhibition Mechanism ◽  
Vitamin K3 ◽  
Vitro Effect

Objective: The present paper investigates the in vitro effect of L-ascorbic acid (vitamin C), menadione sodium bisulfate (vitamin K3), and folic acid on purified lactoperoxidase (LPO). Methods: This enzyme was purified from bovine milk by Amberlite CG 50 resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. Results: Rz (A412/A280) value for the purified LPO was found to be 0.8. Lactoperoxidase was purified 20.45-fold with a yield of 28.8 %. Purity of enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method and a single band was observed. All tested vitamins caused inhibition of the enzyme activity and displayed a competitive type of inhibition mechanism. IC50 values of these three vitamins were 2.03 µM, 0.025 mM, and 0.0925 mM, and the Ki constants were 0.508±0.257 µM, 0.0107±0.0044 mM, and 0.0218±0.0019 mM respectively. Conclusion: The vitamins discussed here displayed inhibition-type competition with LPO enzyme at varying concentrations. Our study showed that L-ascorbic acid exhibited a much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than other vitamins tested.

scholarly journals Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2682-2687 ◽  
Author(s):  
PA Ory ◽  
MR Clark ◽  
AS Talhouk ◽  
IM Goldstein
Keyword(s):  
Molecular Mass ◽  
Cho Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Fc Receptor ◽  
Apparent Molecular Mass ◽  
Human Neutrophils ◽  
Rabbit Reticulocyte Lysate System

Abstract Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.

scholarly journals Purification and properties of a proteolytic enzyme from the cercariae of the human trematode parasite Schistosoma mansoni

1982 ◽  
Vol 201 (1) ◽  
pp. 137-144 ◽  
Author(s):  
W J Landsperger ◽  
M A Stirewalt ◽  
M H Dresden
Keyword(s):  
Amino Acid ◽  
Schistosoma Mansoni ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Isoelectric Point ◽  
Proteolytic Enzymes ◽  
Sodium Dodecyl ◽  
Skin Penetration ◽  
Cathepsin G ◽  
Trematode Parasite

Skin penetration by the cercarial stage of the human trematode parasite Schistosoma mansoni is mediated by the secretion of proteolytic enzymes able to digest components of mammalian connective tissues. In the present study the purification of these proteinases from cercarial homogenates is reported. The major proteinase species has a mol. wt. of approx. 25 000 and exists in monomeric form as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This proteinase has an isoelectric point of 6.0. Studies presented here, with a variety of substrates and inhibitors, confirm previous claims that these proteinases belong to the serine class, and, in addition, suggest that they resemble the vertebrate chymotrypsins rather than trypsins or elastases. However, the amino acid composition of the cercarial proteinase differs significantly from bovine chymotrypsin and from the human leucocyte chymotrypsin-like cathepsin G. The amino-acid-composition differences between these proteinases are consistent with their differences in isoelectric point. In order to obtain an insight into the role of the proteinase in skin penetration, its activity on cartilage proteoglycan monomers and on the isolated peptide backbone of proteoglycan was studied. The results of the present study indicate that the cercarial enzyme catalyses a limited specific digestion of the peptide core.

CD18 integrin and CD54-dependent neutrophil adhesion to cytokine-stimulated human hepatocytes

1997 ◽  
Vol 272 (3) ◽  
pp. G408-G416 ◽  
Author(s):  
A. R. Nagendra ◽  
J. K. Mickelson ◽  
C. W. Smith
Keyword(s):  
Cell Injury ◽  
Proteolytic Enzymes ◽  
Parenchymal Cell ◽  
Intercellular Adhesion Molecule ◽  
Human Neutrophils ◽  
Lymphocyte Function ◽  
Intercellular Adhesion Molecule 1 ◽  
Necrosis Factor Alpha ◽  
Ifn Gamma

We investigated the hypothesis that CD54 (intercellular adhesion molecule-1) expressed on hepatocytes will support beta2-integrin (CD18)-dependent adhesion of neutrophils. An in vitro model using C3A cells (a human hepatoblastoma cell line exhibiting many characteristics of normal hepatocytes) and human neutrophils was utilized. C3A cells were stimulated with interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, or interferon-gamma (IFN-gamma) for 24 h to induce expression of CD54, and adhesion of neutrophils was found to be markedly increased. Detailed studies with IFN-gamma-stimulated (100 U/ml) C3A cells revealed that this adhesion involved CD11a/CD18 [lymphocyte function-associated antigen-1 (LFA-1)] and CD54 and was dependent on low levels of IL-8 produced by the stimulated hepatocytes. Addition of higher concentrations of chemotactic factor (e.g., IL-8) further augmented adhesion and recruited contributions of CD11b/CD18 (Mac-1). In contrast to LFA-1, Mac-1 appeared to recognize a CD54-independent ligand constitutively expressed on the hepatocytes. Such close apposition of neutrophils to hepatocytes may increase the potential for parenchymal cell injury by providing a short distance through which cytotoxic factors such as reactive oxygen or proteolytic enzymes could act.

Transformation of creatine kinase-BB isoenzyme in vitro: effect of carboxylic acids, thiols, pH, cations, and chelators.

1982 ◽  
Vol 28 (12) ◽  
pp. 2414-2417 ◽  
Author(s):  
J A Lott ◽  
J W Heinz
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Carboxylic Acids ◽  
Alkaline Ph ◽  
Creatine Kinase Isoenzyme ◽  
Creatine Kinase Bb ◽  
Vitro Effect ◽  
Dialyzed Serum

Abstract Creatine kinase isoenzyme BB from rat brain was incubated with serum, dialyzed serum, or plasma, with added carboxylic acids, thiols, buffers, or cations. It was found to be very unstable at 37 degrees C under all these conditions, being transformed to CK-BB', a form that migrates like CK-MB on agarose electrophoretic plates. This transformation is enhanced at alkaline pH and, especially, by Zn2+. CK-BB' is quite stable, and probably results from binding of cations to CK-BB, because the transformation is prevented by EDTA and citrate.

scholarly journals In vitro effect of silver nanoparticles on creatine kinase activity

2009 ◽  
Vol 20 (8) ◽  
pp. 1556-1560 ◽  
Author(s):  
Marcos Marques da Silva Paula ◽  
Cláudio Sérgio da Costa ◽  
Mario César Baldin ◽  
Giselli Scaini ◽  
Gislaine Tezza Rezin ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Creatine Kinase ◽  
Kinase Activity ◽  
Creatine Kinase Activity ◽  
Vitro Effect

scholarly journals Defensin-rich dense granules of human neutrophils

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 757-765 ◽  
Author(s):  
WG Rice ◽  
T Ganz ◽  
JM Jr Kinkade ◽  
ME Selsted ◽  
RI Lehrer ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Uranyl Acetate ◽  
Human Neutrophils ◽  
Total Acid ◽  
Density Fractions ◽  
Dense Granules ◽  
Blood Neutrophils

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

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