scholarly journals Human bronchial leucocyte proteinase inhibitor. Rapid isolation and kinetic analysis with human leucocyte proteinases

1985 ◽  
Vol 225 (2) ◽  
pp. 463-472 ◽  
Author(s):  
C E Smith ◽  
D A Johnson
Keyword(s):  
Proteinase Inhibitor ◽  
Dissociation Constants ◽  
Gel Filtration ◽  
Single Step ◽  
Cathepsin G ◽  
Polymorphonuclear Leucocytes ◽  
Bovine Trypsin ◽  
Leucocyte Elastase ◽  
Equilibrium Dissociation Constants

Bronchial leucocyte proteinase inhibitor (BLPI) is an 11 000 Mr protein found in human mucous secretions. This inhibitor apparently controls the serine proteinases elastase and cathepsin G, released from extravascular polymorphonuclear leucocytes. A simple, single-step chromatographic procedure for the isolation of BLPI based on its affinity for chymotrypsin was developed. The purified inhibitor was homogeneous by electrophoresis and gel filtration. Amino acid analyses were in close agreement with previous reports, and showed BLPI to be rich in proline and cystine, but lacking histidine. We have further characterized the role of BLPI with respect to human leucocyte elastase and cathepsin G by close examination of the kinetic parameters. Additionally, we have determined the kinetics of association (kon) and dissociation (koff) for BLPI with bovine trypsin and chymotrypsin. Equilibrium dissociation constants (Ki) of 1.87 × 10(-10) M, 4.18 × 10(-9) M, 8.28 × 10(-9) M and 2.63 × 10(-8) M were obtained for human leucocyte elastase, cathepsin G, bovine trypsin and chymotrypsin, respectively. These results are discussed with respect to BLPI's possible function in vivo and its role relative to other inhibitors in bronchial secretions.

scholarly journals Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

2004 ◽  
Vol 186 (18) ◽  
pp. 6254-6264 ◽  
Author(s):  
Peggy Freede ◽  
Sabine Brantl
Keyword(s):  
Binding Affinity ◽  
Copy Number ◽  
Dissociation Constants ◽  
Mobility Shift ◽  
Copy Numbers ◽  
Maximal Binding ◽  
Equilibrium Dissociation Constants ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5′-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 μM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3′ positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

scholarly journals Purification of a serine-proteinase inhibitor from human articular cartilage. Identity with the acid-stable proteinase inhibitor of mucous secretions

1991 ◽  
Vol 274 (1) ◽  
pp. 269-273 ◽  
Author(s):  
B Böhm ◽  
R Deutzmann ◽  
H Burkhardt
Keyword(s):  
Articular Cartilage ◽  
Proteinase Inhibitor ◽  
Serine Proteinase ◽  
Cathepsin G ◽  
Human Articular Cartilage ◽  
Serine Proteinase Inhibitor ◽  
Inhibitor Molecule ◽  
Leucocyte Elastase ◽  
Sds Page

An inhibitor of the serine proteinases human leucocyte elastase (EC 3.4.21.37), of cathepsin G (EC 3.4.21.20) and of trypsin (EC 3.4.21.4) has been purified from human articular cartilage. The apparent Mr of the cationic (pI greater than 10) protein was determined to 15,000 by SDS/PAGE. It was shown to cross-react in Western blot with a specific antibody to a recombinant-derived serine-proteinase inhibitor of human mucous secretions. Identity of both inhibitors is indicated by the determination of the N-terminal amino acid sequence of the cartilage-derived serine-proteinase inhibitor. In all 24 residues the cartilage inhibitor was shown to be identical with the human secretory leucocyte proteinase inhibitor (‘SLPI’). The inhibitor molecule may play a crucial role in the protection of cartilage matrix proteins against proteolytic attack.

scholarly journals Modification of the tandem reactive centres of human inter-α-trypsin inhibitor with butanedione and cis-dichlorodiammineplatinum(II)

1988 ◽  
Vol 254 (1) ◽  
pp. 171-178 ◽  
Author(s):  
M W Swaim ◽  
S V Pizzo
Keyword(s):  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Proteinase Inhibitor ◽  
Complete Loss ◽  
Cathepsin G ◽  
Partial Loss ◽  
Inhibitory Specificity ◽  
Region Sequence ◽  
Leucocyte Elastase ◽  
Kunitz Type

Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.

scholarly journals Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

2020 ◽  
Vol 21 (1) ◽  
pp. 310 ◽  
Author(s):  
Ema Romão ◽  
Ahmet Krasniqi ◽  
Laila Maes ◽  
Camille Vandenbrande ◽  
Yann G.-J. Sterckx ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dissociation Constants ◽  
Background Signal ◽  
Imaging Results ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Acute Myeloid

Nanobodies (Nbs) are the smallest antigen-binding, single domain fragments derived from heavy-chain-only antibodies from Camelidae. Among the several advantages over conventional monoclonal antibodies, their small size (12–15 kDa) allows them to extravasate rapidly, to show improved tissue penetration, and to clear rapidly from blood, which are important characteristics for cancer imaging and targeted radiotherapy. Herein, we identified Nbs against CD33, a marker for acute myeloid leukemia (AML). A total of 12 Nbs were generated against recombinant CD33 protein, out of which six bound natively CD33 protein, expressed on the surface of acute myeloid leukemia THP-1 cells. The equilibrium dissociation constants (KD) of these six Nbs and CD33 range from 4 to 270 nM, and their melting temperature (Tm) varies between 52.67 and 67.80 °C. None of these Nbs showed leukemogenicity activity in vitro. The selected six candidates were radiolabeled with 99mTc, and their biodistribution was evaluated in THP-1-tumor-bearing mice. The imaging results demonstrated the fast tumor-targeting capacity of the Nbs in vivo. Among the anti-CD33 Nbs, Nb_7 showed the highest tumor uptake (2.53 ± 0.69 % injected activity per gram (IA/g), with low background signal, except in the kidneys and bladder. Overall, Nb_7 exhibits the best characteristics to be used as an anti-CD33 targeting vehicle for future diagnostic or therapeutic applications.

scholarly journals Synthesis and release of platelet-activating factor is inhibited by plasma alpha 1-proteinase inhibitor or alpha 1-antichymotrypsin and is stimulated by proteinases.

1988 ◽  
Vol 168 (4) ◽  
pp. 1293-1306 ◽  
Author(s):  
G Camussi ◽  
C Tetta ◽  
F Bussolino ◽  
C Baglioni
Keyword(s):  
Endothelial Cells ◽  
Human Plasma ◽  
Proteinase Inhibitor ◽  
Vascular Endothelial Cells ◽  
Close Contact ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Cathepsin G ◽  
Vascular Endothelial ◽  
Low Concentrations

TNF and IL-1 stimulate the synthesis and release of platelet-activating factor (PAF) by neutrophils and vascular endothelial cells. Serum inhibits PAF production even after inactivation of an acetylhydrolase that degrades PAF. Human plasma was fractionated by gel filtration chromatography, and two inhibitory fractions were detected, one containing PAF-acetylhydrolase activity and the other alpha 1-proteinase inhibitor. Low concentrations of this antiproteinase and of human plasma alpha 1-antichymotrypsin inhibited TNF-induced PAF synthesis in neutrophils, macrophages, and vascular endothelial cells. Both antiproteinases also inhibited PAF production stimulated by phagocytosis in macrophages and induced with IL-1 in neutrophils or with TNF in vascular endothelial cells. These results suggest that a proteinase activated on the plasma membrane or secreted by these cells is involved in promoting PAF synthesis. Indeed, addition of elastase to macrophages, neutrophils, and endothelial cells stimulated synthesis and release of PAF much faster than TNF. A similar stimulation was observed in incubations with cathepsin G. To identify a proteinase activated in TNF-treated cells, neutrophils and endothelial cells were incubated with specific chloromethyl ketone inhibitors of elastase and cathepsin G. Synthesis of PAF was significantly inhibited by low concentrations of the cathepsin G inhibitor. The finding that antiproteinases are inhibitory at concentrations 100-fold lower than those present in plasma raises questions as to the ability of TNF and IL-1 to stimulate neutrophils in circulation or endothelial cells to synthesize PAF. We propose that PAF production is limited to zones of close contact between cells, which exclude antiproteinases.

Degradation in vivo of articular cartilage in rheumatoid arthritis by leucocyte elastase from polymorphonuclear leucocytes

1981 ◽  
Vol 1 (3) ◽  
pp. 121-130 ◽  
Author(s):  
M. Velvart ◽  
K. Fehr ◽  
A. Baici ◽  
G. Sommermeyer ◽  
M. Kn�pfel ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Articular Cartilage ◽  
Polymorphonuclear Leucocytes ◽  
Leucocyte Elastase

scholarly journals Asprosin Neutralizing Antibodies as a Treatment for Metabolic Syndrome

2020 ◽  
Author(s):  
Ila Mishra ◽  
Clemens Duerrschmid ◽  
Zhiqiang Ku ◽  
Wei Xie ◽  
Elizabeth Sabath Silva ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Blood Glucose ◽  
Neutralizing Antibodies ◽  
Dissociation Constants ◽  
Drug Application ◽  
Distinct Species ◽  
Pharmacologic Therapy ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation

AbstractRecently, we discovered a new glucogenic and centrally-acting orexigenic hormone – asprosin. Asprosin is elevated in metabolic syndrome (MS) patients, and importantly, its genetic loss results in reduced appetite, leanness and robust insulin sensitivity, leading to protection from MS. Here we demonstrate that anti-asprosin monoclonal antibodies (mAbs) are a dual-effect pharmacologic therapy that targets the two key pillars of MS – over-nutrition and the blood glucose burden. Anti-asprosin mAbs from three distinct species lowered appetite and body weight, and improved blood glucose in a dose-dependent and epitope-agnostic fashion in three independent MS mouse models, with an IC50 of ∼1.5 mg/kg. In addition, mAb treatment ameliorated MS associated dyslipidemia and hepatic dysfunction. The mAbs displayed half-life of over 3 days in vivo, with equilibrium dissociation-constants in picomolar to low nanomolar range. This evidence paves the way for further development towards an investigational new drug application and subsequent human trials for treatment of MS, a defining physical ailment of our time.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Defibrotide Inhibits Platelet Activation by Cathepsin G Released From Stimulated Polymorphonuclear Leukocytes

1992 ◽  
Vol 67 (06) ◽  
pp. 660-664 ◽  
Author(s):  
Virgilio Evangelista ◽  
Paola Piccardoni ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Platelet Activation ◽  
Polymorphonuclear Leukocytes ◽  
Direct Interaction ◽  
Cathepsin G ◽  
In Vivo Test ◽  
Cell Functions ◽  
Test Models ◽  
Antithrombotic Effects

SummaryDefibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

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