scholarly journals THE PARTICULATE HYDROLASES OF MACROPHAGES

1963 ◽  
Vol 118 (6) ◽  
pp. 991-1008 ◽  
Author(s):  
Zanvil A. Cohn ◽  
Edith Wiener
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Intact Cell ◽  
Gradient Centrifugation ◽  
Hydrolytic Enzymes ◽  
Neutral Red ◽  
Mechanical Trauma ◽  
Freezing And Thawing ◽  
Alveolar Cells ◽  
Cell Content

The contents of selected hydrolytic enzymes of oil-induced peritoneal, normal alveolar, and BCG-induced alveolar macrophages have been studied. On a per cell or nitrogen basis the normal alveolar cells contained considerably more acid phosphatase, cathepsin, acid ribonuclease, lysozyme, and lipase than peritoneal cells. The BCG-induced alveolar macrophage exhibited increased levels of acid phosphatase, lysozyme, and lipase as compared to alveolar macrophages from unstimulated rabbits. The morphological differences between these cells was discussed and electron micrographs of the BCG-induced macrophage presented. Fractionation of the BCG-induced macrophage by differential centrifugation showed that 60 to 80 per cent of the total cell content of acid phosphatase, cathepsin, beta glucuronidase, acid ribonuclease, acid deoxyribonuclease, aryl sulfatase, lysozyme, and lipase were localized in a postnuclear fraction which sedimented at 15,000 g. This fraction also contained the majority of the mitochondria as evidenced by its content of cytochrome oxidase. Non-specific esterase was not localized to this fraction. A separation of the hydrolase-containing particles and mitochondria was achieved by isopycnic sucrose gradient centrifugation. Under the conditions employed, the mitochondria distributed at densities of 1.19 to 1.20, whereas the hydrolase particles sedimented to a density of 1.26 to 1.27. Each of the hydrolases including acid phosphatase, beta glucuronidase, cathepsin, lysozyme, and acid ribonuclease exhibited maximum activities in the same gradient fraction. The isolated granules exhibited enzymatic latency, and activation could be achieved by cycles of freezing and thawing or surface active agents. The majority of each of the hydrolytic enzymes could be liberated in a non-particulate form by mechanical trauma. Macrophages which had been stained supravitally with neutral red were fractionated by differential and gradient centrifugation. More than 70 per cent of the dye could be recovered in the particulate hydrolase fraction. The isolated, stained granules resembled those seen in the intact cell.

scholarly journals THE PARTICULATE HYDROLASES OF MACROPHAGES

1963 ◽  
Vol 118 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Zanvil A. Cohn ◽  
Edith Wiener
Keyword(s):  
Acid Phosphatase ◽  
Yeast Cell ◽  
Cell Walls ◽  
Hydrolytic Enzymes ◽  
Neutral Red ◽  
Biochemical Properties ◽  
Extracellular Medium ◽  
Cell Content ◽  
Prolonged Incubation

The influence of phagocytosis on the morphological and biochemical properties of macrophage hydrolase-containing granules has been studied in vitro. Following the uptake of large numbers of heat-killed bacteria, an intracellular rearrangement of hydrolytic enzymes occurred. This was associated with the solubilization of 50 to 60 per cent of the total cell content of acid phosphatase, cathepsin, lysozyme, beta glucuronidase, acid ribonuclease, and acid desoxyribonuclease and with a corresponding decrease in granule-bound enzyme. With more prolonged incubation the majority of the soluble intracellular pool of acid ribonuclease and lysozyme was lost to the extracellular medium. No change in the total content of any of the hydrolases was noted during 180 minutes of incubation in vitro. The morphological fate of the granules was studied by a histochemical method for acid phosphatase. After the phagocytosis of yeast cell walls there was a disappearance of acid phosphatase-positive granules and an accumulation of reaction product about the ingested particle. Experiments employing macrophages which were supravitally stained with neutral red also demonstrated the loss of neutral red-positive granules and the accumulation of the dye about the yeast cell walls. These results strongly suggest that lysis of macrophage granules occurs following phagocytosis and that a portion of the granule contents are then resegregated within the newly formed phagocytic vacuole.

scholarly journals LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

1973 ◽  
Vol 56 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Richard C. Franson ◽  
Moseley Waite
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Sucrose Gradient Centrifugation ◽  
Control Distribution ◽  
Specific Activities ◽  
Selective Increase ◽  
Phospholipases A

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

scholarly journals ISOZYMES OF ACID PHOSPHATASE IN NORMAL AND CALMETTE-GUÉRIN BACILLUS-INDUCED RABBIT ALVEOLAR MACROPHAGES

1968 ◽  
Vol 128 (5) ◽  
pp. 1031-1048 ◽  
Author(s):  
S. G. Axline
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Alveolar Macrophages ◽  
Acid Phosphatase Activity ◽  
Enzyme Inhibitors ◽  
Freezing And Thawing ◽  
Ph Optimum ◽  
Lysosomal Fraction ◽  
Repeated Freezing ◽  
Major Acid

The acid phosphatase activity of normal alveolar and BCG-induced alveolar macrophages has been examined. Five electrophoretically distinct forms of acid phosphatase have been identified in both normal and BCG-induced macrophages. The acid phosphatases can be divided into two major categories. One category, containing four distinct forms, is readily solubilized after repeated freezing and thawing or mechanical disruption The second category, containing one form, is firmly bound to the lysosomal membrane and can be solubilized by treatment of the lysosomal fraction with Triton X-100. The Triton-extractable acid phosphatase and the predominant aqueous soluble acid phosphatase have been shown to differ in the degree of membrane binding, in solubility, in net charge, and in molecular weight. The two pre-dominant phosphatases possess identical pH optimum and do not differ in response to enzyme inhibitors. BCG stimulation has been shown to result in a nearly twofold increase in acid phosphatase activity. A nearly proportionate increase in the major acid phosphatase forms has been observed.

The blood leukocytes of Bufo marinus: a light, phase-contrast, and histochemical study

1987 ◽  
Vol 65 (6) ◽  
pp. 1445-1453 ◽  
Author(s):  
M. Samuel Cannon ◽  
H. W. Sampson ◽  
E. D. Kapes
Keyword(s):  
Acid Phosphatase ◽  
Phase Contrast ◽  
Periodic Acid ◽  
Hydrolytic Enzymes ◽  
Neutral Red ◽  
Bufo Marinus ◽  
Oxidative Enzymes ◽  
Blood Leukocytes ◽  
Periodic Acid Schiff ◽  
Aerobic And Anaerobic

Blood leukocytes of Bufo marinus were studied by light and phase-contrast microscopy and histochemical techniques for the localization of glycogen, lipids, several basic proteins, and a number of hydrolytic and oxidative enzymes. The hydrolytic enzymes occurred in varying amounts in neutrophils, eosinophils, lymphocytes, and monocytes; neutrophils were the only leukocytes to demonstrate alkaline phosphatase activity, while β-glucuronidase was only seen in lymphocytes, and aryl-sulfatase was not observed in any leukocytes. Periodic acid – Schiff (PAS) positive granules also occurred in varying amounts in leukocytes. Slight lipid activity was only seen in neutrophils, while arginine, and (or) lysine, and tyrosine reactivity was only observed in eosinophils. The appearance and histochemical reactivity of acid phosphatase granules in neutrophils corresponded closely with the appearance and number of specific neutrophilic granules seen in Wright–Giemsa preparations and with the PAS-positive granules. Small lymphocytes were myeloperoxidase (peroxidase) negative; β-glucuronidase, acid phosphatase, and PAS-positive granules corresponded to neutral red granules seen in supravital films. The oxidative enzymes also occurred in differing amounts in leukocytes, but strongly suggested that the leukocytes of Bufo marinus are capable of some degree of aerobic and anaerobic metabolism.

The Demonstration of Lysosomes in Mouse Skin

1964 ◽  
Vol s3-105 (69) ◽  
pp. 73-78
Author(s):  
J. V. DIENGDOH
Keyword(s):  
Acid Phosphatase ◽  
Biochemical Characterization ◽  
Biochemical Study ◽  
Hydrolytic Enzymes ◽  
Mouse Skin ◽  
Freezing And Thawing ◽  
Sectioning Method ◽  
Repeated Freezing ◽  
Triton X

Lysosomes, as demonstrated biochemically in the liver, are subcellular particles containing a group of hydrolytic enzymes enclosed by a membrane-like barrier. They are apparently inactive in the normal state, but when subjected to various forms of injurious treatments the enzymes associated with them are released. The existence of lysosomes in skin is demonstrated in the present communication. The resistance of this tissue to homogenization makes the biochemical study of lysosomes very difficult. Yet, by the application of histochemical methods to sections of skin, it has been possible to use the same criteria as would be employed in the biochemical characterization of these organelles. By using the controlled-temperature freezing-sectioning method it has been possible to obtain frozen sections in which cytoplasmic particles could be demonstrated which were enzymically inactive for acid phosphatase until the sections were subjected to such injurious treatments as heat, repeated freezing and thawing, hypotonic solutions, distilled water, and ‘triton-X-100’. Since the subcellular particles demonstrated behaved in the same manner as lysosomes prepared biochemically from liver, it is concluded that the cytoplasmic organelles staining for acid phosphatase in mouse skin are lysosomes as biochemically defined.

The blood leukocytes of Bufo alvarius: a light, phase-contrast, and histochemical study

1979 ◽  
Vol 57 (2) ◽  
pp. 314-322 ◽  
Author(s):  
M. Samuel Cannon ◽  
A. M. Cannon
Keyword(s):  
Acid Phosphatase ◽  
Phase Contrast ◽  
Periodic Acid ◽  
Hydrolytic Enzymes ◽  
Neutral Red ◽  
Nonspecific Esterase ◽  
Alkaline Phosphatases ◽  
Blood Leukocytes ◽  
Periodic Acid Schiff ◽  
Aryl Sulfatase

Blood leukocytes of Bufo alvarius were studied by light and phase-contrast microscopy and histochemical techniques for the localization of glycogen and several hydrolytic enzymes, i.e., acid and alkaline phosphatases, nonspecific esterase, beta-glucuronidase, aryl-sulfatase, and myeloperoxidase (peroxidase). Neutrophils were the only leukocytes to demonstrate alkaline phosphatase activity, while beta-glucuronidase and aryl-sulfatase were not observed in any leukocytes. Periodic acid – Schiff (PAS) positive granules and granules containing hydrolytic enzymes occurred in varying amounts in leukocytes. In eosinophils, most glycogen was associated with smaller granules, while the larger refractile granules were PAS negative. Small lymphocytes were myeloperoxidase (peroxidase) negative. The present study agrees with previous investigations in mammals which indicate that specific granules in granulocytes may be PAS positive as well as contain one or more hydrolytic enzymes. In small lymphocytes of B. alvarius, PAS positive and acid phosphatase positive granules correspond to neutral red granules seen in supravital films. Furthermore, the appearance and histochemical reactivity of acid phosphatase granules in mature neutrophils, metamyelocytes, and late myelocytes correspond closely with the appearance and number of specific neutrophilic granules seen in Wright–Giemsa preparations and with PAS positive granules.

Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity

1998 ◽  
Vol 76 (7-8) ◽  
pp. 721-727 ◽  
Author(s):  
M W Bolt ◽  
W J Racz ◽  
J F Brien ◽  
T M Bray ◽  
T E Massey
Keyword(s):  
Alveolar Macrophages ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Clara Cell ◽  
Blue Dye ◽  
Alveolar Type ◽  
Specific Cell ◽  
Type Ii ◽  
Clara Cells

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 µM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 µM AM. Based on a viability index ((viability of AM-treated cells ÷ viability of controls) × 100%), the Clara cell fraction was significantly (p < 0.05) more susceptible than all of the other cell types to 50 µM AM. However, AM cytotoxicity was greatest (p < 0.05) in alveolar macrophages following incubation with 100 or 200 µM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 µM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 µM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.Key words: amiodarone, susceptibility, alveolar macrophage, accumulation.

scholarly journals Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor

1985 ◽  
Vol 228 (1) ◽  
pp. 111-117 ◽  
Author(s):  
E Davies Jones ◽  
F A Hashim ◽  
Y Kajita ◽  
F M Creagh ◽  
P R Buckland ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Thyrotropin Receptor ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
A Subunit

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves‘ serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes’ radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

Enhancing Effect of Surfactant and Protein on Hydrolysis of Thymolphthalein Monophosphate by Purified Prostatic Acid Phosphatase

1975 ◽  
Vol 21 (12) ◽  
pp. 1761-1765 ◽  
Author(s):  
Andras G Foti ◽  
Harvey Herschman ◽  
J Fenimore Cooper ◽  
Hedi imFeld
Keyword(s):  
Acid Phosphatase ◽  
Serum Proteins ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Prostatic Acid Phosphatase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Brij 35 ◽  
Hydrolysis Of ◽  
Protein Bovine Serum Albumin

Abstract Purified prostatic acid phosphatase catalyzes the hydrolysis of thymolphthalein monophosphate 10-fold faster if an optimal concentration of Brij 35 (a wetting agent) or protein (bovine serum albumin or human serum proteins) is present. Results of gel filtration, dialysis, and sucrose density-gradient centrifugation analysis suggest that the substrate must combine with detergent or protein before the enzyme can catalyze its hydrolysis.

scholarly journals MACROPHAGE PLASMA MEMBRANES

1971 ◽  
Vol 133 (4) ◽  
pp. 785-806 ◽  
Author(s):  
Ralph L. Nachman ◽  
Barbara Ferris ◽  
James G. Hirsch
Keyword(s):  
Alveolar Macrophages ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Membrane Preparation ◽  
Polystyrene Spheres ◽  
Protein Patterns ◽  
Large Numbers ◽  
Structural Preservation ◽  
High Degree

Plasma membranes have been isolated from pure populations of rabbit alveolar macrophages which were swollen in water, fixed briefly with glutaraldehyde, disrupted by Dounce homogenization, and separated by sucrose gradient centrifugation. The recovered membranes exhibited good structural preservation and enzymatic activity; both morphologic and biochemical evidence indicated a high degree of purity (&gt;90%) of the membrane preparation. Interiorized plasma membranes were also prepared without exposure to glutaraldehyde from phagocytic vacuoles recovered from alveolar macrophages which had ingested large numbers of polystyrene spheres. These membranes were contaminated with lysosomal constituents, but they were nevertheless of value for comparison to the "pure" membranes isolated by the glutaraldehyde procedure. Acrylamide gel electrophoresis of the solubilized plasma membranes and phagolysosomal membranes revealed similar protein patterns, with seven to nine individual components ranging in molecular weight from 70,000 to 140,000. The two most rapidly migrating components gave positive reactions for lipid as well as protein. A band containing carbohydrate was detected near the origin of the plasma membrane gels. Antisera were made by injecting guinea pigs with the purified rabbit alveolar macrophage plasma membranes. Gel diffusion and immunoelectrophoretic study of these antisera established the presence of rabbit immunoglobulin G and of one or two other antigenic constituents in the membrane preparation.

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