Neutral Protease from the Polymorphonuclear Leucocytes of Human Rheumatoid Synovial Fluid

1974 ◽  
Vol 47 (5) ◽  
pp. 403-414
Author(s):  
R. H. Pryce-Jones ◽  
J. Saklatvala ◽  
G. C. Wood
Keyword(s):  
Ionic Strength ◽  
Synovial Fluid ◽  
Protease Activity ◽  
Degradation Products ◽  
Neutral Protease ◽  
Polymorphonuclear Leucocytes ◽  
Bovine Articular Cartilage ◽  
Rheumatoid Synovial Fluid ◽  
Synovial Fluids ◽  
Low Ionic Strength

1. The cartilage-proteoglycan-degrading activity of synovial fluid cells from rheumatoid patients is primarily due to neutral protease activity; hyaluronidase does not contribute significantly. Evidence for these conclusions is adduced from electrophoresis of degradation products, from comparison of the proteoglycan-degrading and protease activities of extracts and from the effects of activators and inhibitors. 2. Most of the neutral protease activity is insoluble in buffer of low ionic strength and may thus be separated from small amounts of other, soluble, proteoglycan-degrading enzymes. This ‘insoluble’ protease dissolves in KCl (1 mol/l) and has appreciable solubility even at physiological ionic strength. 3. The protease has optimum activity between pH 7.5 and pH 10, with little or no activity below pH 6.0. 4. The enzyme hydrolyses a number of substrates including elastin and two synthetic substrates for elastase. Hydrolysis of the latter is inhibited by dilutions of synovial fluids and serum and it thus differs from the elastase-like esterase activity of synovial fluid and serum. 5. The ‘insoluble’ enzyme fraction caused loss of staining and release of hexuronate from slices of bovine articular cartilage at salt concentrations near physiological and these effects were almost completely inhibited by cell-free synovial fluids. 6. The properties of the synovial fluid cell neutral protease are compared with those of the similar enzyme activity found in granulocytes from blood and it is concluded that the synovial fluid enzyme is probably derived from granulocytes.

scholarly journals Chondromucoprotein-degrading neutral protease activity in rheumatoid synovial fluid.

1971 ◽  
Vol 30 (1) ◽  
pp. 73-77 ◽  
Author(s):  
G C Wood ◽  
R H Pryce-Jones ◽  
D D White ◽  
G Nuki
Keyword(s):  
Synovial Fluid ◽  
Protease Activity ◽  
Neutral Protease ◽  
Rheumatoid Synovial Fluid

scholarly journals Characterization of a neutral protease from lysosomes of rabbit polymorphonuclear leucocytes

1971 ◽  
Vol 123 (4) ◽  
pp. 559-569 ◽  
Author(s):  
Philip Davies ◽  
Giuseppe A. Rita ◽  
Kathrin Krakauer ◽  
Gerald Weissmann
Keyword(s):  
Ionic Strength ◽  
Protease Activity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Artificial Substrates ◽  
Neutral Protease ◽  
Polymorphonuclear Leucocytes ◽  
Whole Cells ◽  
Bean Trypsin Inhibitor

1. The subcellular distribution has been investigated of a protease from rabbit polymorphonuclear leucocytes, obtained from peritoneal exudates. The enzyme, optimally active between pH7.0 and 7.5, hydrolyses histone but not haemoglobin, sediments almost exclusively with a granule fraction rich in other lysosomal enzymes, and is latent until the granules are disrupted by various means. 2. Enzymic analysis of specific and azurophilic granules separated by zonal centrifugation showed that neutral protease activity was confined to fractions rich in enzymes characteristic of azurophile granules. 3. Recovery of neutral protease activity from subcellular fractions was several times greater than that found in whole cells. This finding was explained by the presence of a potent inhibitor of the enzyme activity in the cytoplasm. 4. The effect of the inhibitor was reversed by increasing ionic strength (up to 2.5m-potassium chloride) and by polyanions such as heparin and dextran sulphate, but not by an uncharged polymer, dextran. 5. The enzyme was also inhibited, to a lesser extent, by 1-chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one, soya-bean trypsin inhibitor and ∈-aminohexanoate (∈-aminocaproate). 6. The granule fractions failed to hydrolyse artificial substrates for trypsin and chymotrypsin. 7. Partial separation of the enzyme was achieved by Sephadex gel filtration at high ionic strength and by isoelectric focusing. The partially separated, activated enzyme showed an approximately 300-fold increase in specific activity over that in whole cells.

Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

An Evaluation of a Plasma Fractionation System

1960 ◽  
Vol 4 (01) ◽  
pp. 031-044
Author(s):  
George Y. Shinowara ◽  
E. Mary Ruth
Keyword(s):  
Biological Activity ◽  
Ionic Strength ◽  
Plasma Proteins ◽  
High Concentration ◽  
Significant Evidence ◽  
Native Proteins ◽  
Mobility Data ◽  
Fractionation Procedure ◽  
The Individual ◽  
Low Ionic Strength

SummaryFour primary fractions comprising at least 97 per cent of the plasma proteins have been critically appraised for evidence of denaturation arising from a low temperature—low ionic strength fractionation system. The results in addition to those referable to the recovery of mass and biological activity include the following: The high solubilities of these fractions at pH 7.3 and low ionic strengths; the compatibility of the electrophoretic and ultracentrifugal data of the individual fractions with those of the original plasma; and the recovery of hemoglobin, not hematin, in fraction III obtained from specimens contaminated with this pigment. However, the most significant evidence for minimum alterations of native proteins was that the S20, w and the electrophoretic mobility data on the physically recombined fractions were identical to those found on whole plasma.The fractionation procedure examined here quantitatively isolates fibrinogen, prothrombin and antithrombin in primary fractions. Results have been obtained demonstrating its significance in other biological systems. These include the following: The finding of 5 S20, w classes in the 4 primary fractions; the occurrence of more than 90 per cent of the plasma gamma globulins in fraction III; the 98 per cent pure albumin in fraction IV; and, finally, the high concentration of beta lipoproteins in fraction II.

scholarly journals A novel procedure for the rapid purification of plastocyanin from pea (Pisum sativum) leaves

1981 ◽  
Vol 193 (1) ◽  
pp. 375-378 ◽  
Author(s):  
A R Ashton ◽  
L E Anderson
Keyword(s):  
Ionic Strength ◽  
Pisum Sativum ◽  
Deae Cellulose ◽  
High Concentrations ◽  
Rapid Purification ◽  
Low Ionic Strength

Plastocyanin is soluble at high concentrations (greater than 3 M) of (NH4)2SO4 but under these conditions will adsorb tightly to unsubstituted Sepharose beads. This observation was utilized to purify plastocyanin from pea (Pisum sativum) in two chromatographic steps. Sepharose-bound plastocyanin was eluted with low-ionic-strength buffer and subsequently purified to homogeneity by DEAE-cellulose chromatography.

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