scholarly journals ASSOCIATION OF THE ALKALINE PHOSPHATASE OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES WITH THE MEMBRANE OF THE SPECIFIC GRANULES

1973 ◽  
Vol 59 (3) ◽  
pp. 696-707 ◽  
Author(s):  
Ursula Bretz ◽  
Marco Baggiolini
Keyword(s):  
Alkaline Phosphatase ◽  
Polymorphonuclear Leukocyte ◽  
Sodium Sulfate ◽  
Polymorphonuclear Leukocytes ◽  
Sodium Deoxycholate ◽  
Soluble Form ◽  
Hexadecyltrimethylammonium Bromide ◽  
Specific Granules ◽  
Granule Membrane ◽  
Triton X

The localization of alkaline phosphatase in the specific granules of rabbit polymorphonuclear leukocytes was investigated. The results obtained suggest very strongly that alkaline phosphatase is a component of the granule membrane. The enzyme remains attached to the membrane upon disruption of the granules by the use of detergents or by hypotonic shock and subsequent extraction with sodium sulfate, and can be isolated together with fragments of the granule membrane by isopycnic equilibration. Treatment of the granules with high amounts of Triton-X-100, sodium deoxycholate, or hexadecyltrimethylammonium bromide releases the enzyme in soluble form. In polymorphonuclear leukocyte homogenates, lysis of the granules is needed in order to render alkaline phosphatase fully accessible to substrates. This suggests that the catalytic site of the enzyme is exposed at the inner face of the granule membrane.

scholarly journals SEQUENTIAL DEGRANULATION OF THE TWO TYPES OF POLYMORPHONUCLEAR LEUKOCYTE GRANULES DURING PHAGOCYTOSIS OF MICROORGANISMS

1973 ◽  
Vol 58 (2) ◽  
pp. 249-264 ◽  
Author(s):  
Dorothy Ford Bainton
Keyword(s):  
Electron Microscopy ◽  
Alkaline Phosphatase ◽  
Polymorphonuclear Leukocyte ◽  
Lysosomal Enzymes ◽  
Marker Enzyme ◽  
Optimal Conditions ◽  
Alkaline Ph ◽  
Enzyme Content ◽  
Specific Granules ◽  
Acid Range

The sequential discharge of neutrophilic polymorphonuclear leukocyte (PMN) granules—azurophils and specifics—was investigated by electron microscopy and cytochemistry. Thus the enzyme content of PMN phagocytic vacuoles was determined at brief intervals after phagocytosis of bacteria, utilizing peroxidase as a marker enzyme for azurophil granules, and alkaline phosphatase for specifics. At 30 s, approximately half the phagocytic vacuoles were reactive for alkaline phosphatase, whereas none contained peroxidase. Peroxidase-containing vacuoles were rarely seen at 1 min, but by 3 min, vacuoles containing both enzymes were consistently present. Alkaline phosphatase was found in both small and large vacuoles, whereas peroxidase was visible only in large ones. By 10 min, very big phagocytic vacuoles containing considerable amounts of reaction product for both enzymes were evident. These observations indicate that the two types of PMN granules discharge in a sequential manner, specific granules fusing with the vacuole before azurophils. In an earlier paper, we reported that the pH of phagocytic vacuoles drops to 6.5 within 3 min and to ∼4 within 7–15 min. Substances known to be present in specific granules (alkaline phosphatase, lysozyme, and lactoferrin) function best at neutral or alkaline pH, whereas most of those contained in azurophil granules (i.e., peroxidase and the lysosomal enzymes) have pH optima in the acid range. Hence the sequence of granule discharge roughly parallels the change in pH, thereby providing optimal conditions for coordinated activity of granule contents.

scholarly journals DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES

1968 ◽  
Vol 39 (2) ◽  
pp. 286-298 ◽  
Author(s):  
Dorothy Ford Bainton ◽  
Marilyn G. Farquhar
Keyword(s):  
Alkaline Phosphatase ◽  
Polymorphonuclear Leukocytes ◽  
Basic Protein ◽  
Developmental Stages ◽  
Hydrolytic Enzymes ◽  
Enzyme Content ◽  
Specific Granules ◽  
Rabbit Bone ◽  
Thiolacetic Acid ◽  
Enzyme Alkaline Phosphatase

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, ß-galactosidase, ß-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.

scholarly journals BIOCHEMICAL AND MORPHOLOGICAL CHARACTERIZATION OF AZUROPHIL AND SPECIFIC GRANULES OF HUMAN NEUTROPHILIC POLYMORPHONUCLEAR LEUKOCYTES

1974 ◽  
Vol 63 (1) ◽  
pp. 251-269 ◽  
Author(s):  
Ursula Bretz ◽  
Marco Baggiolini
Keyword(s):  
Alkaline Phosphatase ◽  
Membrane Fraction ◽  
Polymorphonuclear Leukocytes ◽  
Morphological Characterization ◽  
Average Density ◽  
Lysosomal Hydrolases ◽  
Specific Granules ◽  
Neutrophilic Polymorphonuclear Leukocytes ◽  
A Minor

Postnuclear supernates from homogenates of purified neutrophil polymorphonuclear leukocytes (PMNs) from human blood were fractionated by zonal sedimentation and isopycnic equilibration in sucrose gradients. The fractions were characterized biochemically by measuring protein content and the activities of eight enzymes. Selected fractions were further analyzed by electron microscopy. In both centrifugation systems, azurophil and specific granules could be resolved almost completely. Azurophil granules sediment three to four times faster than the specifics and have an average density of 1.23. They contain all the peroxidase of the cells, large portions of four lysosomal hydrolases, and about half of the total lysozyme, and therefore appear to be, in biochemical terms, very similar to the azurophil granules of rabbit PMNs. The specific granules, which have an average density of 1.19, contain the remaining half of the lysozyme but appear to be free of the other components of the azurophil granules, and of alkaline phosphatase. Isopycnic equilibration disclosed a minor lysosomal population, which strongly overlaps the specific granules, and made possible the identification of a membrane-fraction which is characterized by the presence of the thiol-sensitive acid 4-nitrophenyl phosphatase and of alkaline phosphatase.

scholarly journals STUDIES ON ISOLATED MEMBRANES OF AZUROPHIL AND SPECIFIC GRANULES FROM RABBIT POLYMORPHONUCLEAR LEUKOCYTES

1972 ◽  
Vol 54 (1) ◽  
pp. 133-140 ◽  
Author(s):  
Ralph Nachman ◽  
James G. Hirsch ◽  
Marco Baggiolini
Keyword(s):  
Polymorphonuclear Leukocyte ◽  
Polymorphonuclear Leukocytes ◽  
Differential Centrifugation ◽  
Polyacrylamide Gels ◽  
Specific Granules ◽  
Ultrastructural Appearance ◽  
Protein Components

Membranes were prepared from rabbit polymorphonuclear leukocyte azurophil and specific granules separated by zonal differential centrifugation. The two types of granule membranes were quite similar in ultrastructural appearance, but they showed distinct differences in cholesterol-phospholipid ratios and in protein components demonstrable in polyacrylamide gels.

scholarly journals ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES

1966 ◽  
Vol 28 (2) ◽  
pp. 277-301 ◽  
Author(s):  
Dorothy Ford Bainton ◽  
Marilyn G. Farquhar
Keyword(s):  
Bone Marrow ◽  
Golgi Complex ◽  
Polymorphonuclear Leukocyte ◽  
Polymorphonuclear Leukocytes ◽  
Dense Core ◽  
Granule Formation ◽  
Specific Granules ◽  
Azurophil Granule ◽  
Rabbit Bone ◽  
Specific Granule

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO4. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (∼800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (∼500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.

scholarly journals Electrophoretic characterization of hepatic alkaline phosphatase released by phosphatidylinositol-specific phospholipase C. A comparison with liver membrane and serum-soluble forms

1985 ◽  
Vol 227 (1) ◽  
pp. 183-189 ◽  
Author(s):  
T Kominami ◽  
A Miki ◽  
Y Ikehara
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Phospholipase C ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Polyacrylamide Gel ◽  
Soluble Form ◽  
Enzyme Preparations

Alkaline phosphatase was solubilized from plasma membrane of rat liver with butanol-ol, bile acids or sodium deoxycholate, and electrophoretically compared with a soluble form in serum which was derived from the liver. The three enzyme preparations from the plasma membrane migrated at the same position on polyacrylamide-gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulphate. The mobility of them, however, was distinctly different from that of the serum-soluble form of the liver-derived alkaline phosphatase. On the other hand, phosphatidylinositol-specific phospholipase C isolated from Bacillus cereus was used to release alkaline phosphatase from plasma membrane. The released alkaline phosphatase was demonstrated to have the same mobility as the serum-soluble form on polyacrylamide-gel electrophoresis in the presence or absence of detergents. The phospholipase C also converted the butan-1-ol-extracted membrane form into the serum-soluble form. The results suggest that release of alkaline phosphatase from the liver into serum is not simply caused by a detergent effect of bile salts, but involves an enzymic hydrolysis of phosphatidylinositol, with which alkaline phosphatase may strongly interact in the membrane.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

scholarly journals Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C

1987 ◽  
Vol 241 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Y Ikehara ◽  
Y Hayashi ◽  
S Ogata ◽  
A Miki ◽  
T Kominami
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Soluble Form ◽  
Microsomal Membranes ◽  
Hydrophobic Nature ◽  
Rat Hepatoma

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.

scholarly journals Formose reaction accelerated in aerosol-OT reverse micelles

2016 ◽  
Vol 12 ◽  
pp. 2663-2667 ◽  
Author(s):  
Makoto Masaoka ◽  
Tomohiro Michitaka ◽  
Akihito Hashidzume
Keyword(s):  
Reverse Micelles ◽  
Water Layer ◽  
Major Product ◽  
Hexadecyltrimethylammonium Bromide ◽  
Interfacial Water ◽  
Formose Reaction ◽  
Aerosol Ot ◽  
Aot Reverse Micelles ◽  
Triton X ◽  
C Nmr

The formose reaction in reverse micelles of aerosol-OT (AOT), triton X-100 (TX), and hexadecyltrimethylammonium bromide (CTAB) was investigated. Time–conversion data have indicated that the interfacial water layer of AOT reverse micelles is a medium that accelerates formation of glycolaldehyde in the formose reaction. The 13C NMR spectra for the products of the formose reaction using formaldehyde-13C as starting material are indicative of the formation of ethylene glycol as a major product.

scholarly journals Effects of leukocyte-derived cathepsin G on platelet membrane glycoprotein Ib-IX and IIb-IIIa complexes: a comparison with thrombin

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2442-2451
Author(s):  
M Molino ◽  
M Di Lallo ◽  
N Martelli ◽  
G de Gaetano ◽  
C Cerletti
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Fluorescein Isothiocyanate ◽  
Adenosine Diphosphate ◽  
Cathepsin G ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Antigenic Sites ◽  
Triton X ◽  
Von Willebrand ◽  
Willebrand Factor

Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC- 1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA- fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb- IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.

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