scholarly journals Localization mechanisms in enzyme cytochemistry studied with alkaline phosphatase-loaded erythrocyte ghosts.

1984 ◽  
Vol 32 (8) ◽  
pp. 815-820 ◽  
Author(s):  
A K Raap ◽  
P Van Duijn
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Salt ◽  
Diffusion Constant ◽  
Theory And Practice ◽  
Minimal Amount ◽  
Erythrocyte Ghosts ◽  
Enzyme Localization ◽  
Enzyme Cytochemistry ◽  
Capture Reaction ◽  
Azo Compound

Erythrocyte ghosts containing varying amounts of alkaline phosphatase were used to study the localization mechanisms of three metal salt and one azo method for this enzyme. For the azo method, the minimal amount of alkaline phosphatase that can be visualized within the ghosts proved only to be limited by the optical properties of the azo compound. In contrast, for the metal salt methods, a certain threshold activity had to be present in the ghosts in order to obtain correct localization of the final reaction product. The localization properties of both azo and metal salt methods conformed to the theories of cytochemical enzyme localization presented to date. By determining the rate constant of the capture reaction and the diffusion constant of the primary product, the localization properties of the azo method could be predicted. Some remaining discrepancies between theory and practice are discussed.

scholarly journals Enzyme-incorporated erythrocyte ghosts: a new model system for quantitative enzyme cytochemistry.

1981 ◽  
Vol 29 (12) ◽  
pp. 1418-1424 ◽  
Author(s):  
A K Raap ◽  
P Van Duijn
Keyword(s):  
Alkaline Phosphatase ◽  
Coupling Agent ◽  
Human Erythrocyte ◽  
Diazonium Salt ◽  
Model System ◽  
Enzymic Activity ◽  
Erythrocyte Ghosts ◽  
Biochemical Activity ◽  
Enzyme Cytochemistry ◽  
Low Levels

The preparation and properties of a new microscopic model system for quantitative enzyme cytochemistry are described. The enzyme to be studied is entrapped in human erythrocyte ghosts by a simple hypotonic procedure. After fixation in suspension the ghosts can be analyzed both biochemically and cytochemically. The system has been tested with alkaline phosphatase. It is demonstrated that an azo method that uses naphthol AS-MX phosphate as substrate and 4-aminodiphenylamine diazonium salt as coupling agent can detect very low levels of enzymic activity. The biochemical activity determinations of alkaline phosphatase loaded erythrocyte ghosts were found to correlate linearly with cytophotometric activity determinations. The possible use of the erythrocyte ghost model system for other cytochemical applications is briefly discussed.

scholarly journals A rapid method for determining diffusion constants in solution

1948 ◽  
Vol 192 (1030) ◽  
pp. 382-402 ◽  
Keyword(s):  
Monochromatic Light ◽  
Diffusion Constant ◽  
Initial Boundary ◽  
Optic Axis ◽  
Theory And Practice ◽  
Dilute Solution ◽  
Sharp Boundary ◽  
Diffusion Constants ◽  
A Cell ◽  
Molecular Substances

A method has been rediscovered, and developed in theory and practice, for optical observation of the earliest stages of diffusion across an initially sharp boundary between a dilute solution and a solvent. It enables the diffusion constant of a monodisperse solute to be measured about fifty times as quickly as by other methods, at lower concentration and possibly with greater accuracy; it should therefore be particularly valuable for the study of high molecular substances. The method is based on the interference pattern which is formed when monochromatic light from a horizontal slit is focused after passing through a cell where diffusion is occurring. The pattern, a set of horizontal bands, contracts towards the optic axis as diffusion proceeds, at a rate from which the diffusion constant can be calculated. By counting the bands in the pattern the refractive increment of the solute can be determined. The sharp initial boundary is obtained by flowing the solution and solvent out through a common narrow horizontal slit. The construction, calibration, and use of the apparatus are described.

scholarly journals A NEW METHOD FOR THE INVESTIGATION OF THE CAPTURE REACTION IN PHOSPHATASE CYTOCHEMISTRY III. EFFECTS OF THE COMPOSITION OF THE INCUBATION MEDIUM ON THE TRAPPING OF PHOSPHATE IONS IN A MODEL SYSTEM

1974 ◽  
Vol 22 (2) ◽  
pp. 110-119 ◽  
Author(s):  
C. J. CORNELISSE ◽  
P. VAN DUIJN
Keyword(s):  
Model System ◽  
Chloride Ions ◽  
Trapping Efficiency ◽  
Lead Salt ◽  
Lead Phosphate ◽  
Enzyme Cytochemistry ◽  
Factors Affecting ◽  
Capture Reaction ◽  
Phosphate Ions ◽  
Phosphatase Cytochemistry

A model system developed for the study of diffusion problems in lead salt enzyme cytochemistry served as a basis for the experiments reported in the present paper. Phosphate leakage, which was investigated in polyacrylamide films during incubation in media containing lead, could be expressed by a graphical parameter related to the average displacement of the phosphate ions prior to the onset of lead phosphate precipitation in the films. This parameter can be used as a measure for the efficiency of the capture reaction in cytochemical reactions in tissue sections. The concentration of lead ions, and to a lesser extent that of the phosphate ions, was found to be critical for the degree of phosphate diffusion in this system. Addition of 0.3% of β-glycerophosphate to the lead medium gave increased phosphate diffusion, and trapping was markedly improved by added chloride ions. Raising of the molarity of the acetate buffer resulted in impaired trapping efficiency. Similar findings were made in a parallel study on the kinetics of lead phosphate precipitation in supersaturated solutions with a nephelometric method. The results of the present investigation indicate that the film model system is highly suitable for the study of factors affecting the rapid immobilization of diffusible products of cytochemical reactions.

DIFFUSION CONSTANT MEASUREMENT IN THEORY AND PRACTICE

1945 ◽  
Vol 46 (5 The Diffusion) ◽  
pp. 309-327 ◽  
Author(s):  
Edward M. Bevilacqua ◽  
Ellen B. Bevilacqua ◽  
Margaret M. Bender ◽  
J. W. Williams
Keyword(s):  
Diffusion Constant ◽  
Theory And Practice ◽  
Constant Measurement

Preliminary Studies on Heterotopic Calcinosis

1970 ◽  
Vol 28 ◽  
pp. 242-243
Author(s):  
J.R. Scott ◽  
R.B. Marshall ◽  
D.K. Roberts
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Connective Tissue ◽  
Cell Population ◽  
Giant Cells ◽  
Tumoral Calcinosis ◽  
Fibrous Connective Tissue ◽  
Enzyme Cytochemistry ◽  
Calcium Salts ◽  
Aryl Sulfatase

Interstitial calcinosis and tumoral calcinosis lesions were studied ultrastructurally and with the use of enzyme cytochemistry. Technics for alkaline phosphatase, acid phosphatase, atpase and aryl sulfatase were utilized after removal of calcium salts with EDTA.Granular elements were seen in both lesions. These granules were seen to lie in cystic spaces of the lesions (Figure 1). Also seen were aggregates of these granules which had apparently calcified (Figure 2). The lesion of tumoral calcinosis had many multinuclear giant cells present peripheral to the cystic spaces with macrophages contributing to the cell population. The lesions all had a pseudocapsule of fibrous connective tissue and “active” connective tissue cells were seen near the cystic space.

Biochemical and Cytochemical Effects of Cytosine Arabinoside (ARA C) and Hydrocortisone on hela cells

1979 ◽  
Vol 65 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Silvia D'Ancona ◽  
Paolo Adami ◽  
Roberto Poltronieri ◽  
Maria Teresa Barberio ◽  
Paolo Rizzotti
Keyword(s):  
Alkaline Phosphatase ◽  
Hela Cells ◽  
Cytosine Arabinoside ◽  
Enzyme Localization ◽  
Cytochemical Technique

The biochemical and cytochemical effects of cytosine arabinoside (Ara C) and of hydrocortisone on HeLa cells in vitro have been studied. In cultures treated with Ara C, a relationship exists between the cytocidal effects of the drug and an increase in alkaline phosphatase. Although hydrocortisone had no influence on the proliferative rhythm, it induced an increase in alkaline phosphatase. Results obtained with the cytochemical technique were evaluated, particularly in relation to enzyme localization.

Ultrastructural localization of alkaline phosphatase activity in human eccrine and apocrine sweat glands.

1995 ◽  
Vol 43 (9) ◽  
pp. 927-932 ◽  
Author(s):  
K Saga ◽  
Y Morimoto
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Membranes ◽  
Ultrastructural Localization ◽  
Sweat Glands ◽  
Secretory Cells ◽  
Electron Microscopic ◽  
Enzyme Cytochemistry ◽  
Alp Activity ◽  
Eccrine Sweat Glands ◽  
Eccrine Sweat

Alkaline phosphatase (ALP) is a membrane-bound enzyme that catalyzes the hydrolysis of inorganic and organic monophosphate esters at alkaline pH. Although the functions of ALP are poorly understood, it is believed to be involved in membrane transport. Because little is known about the functions and distribution of ALP in the sweat glands, we studied the localization of ALP in human sweat glands with light and electron microscopic enzyme cytochemistry. In eccrine sweat glands, ALP was restricted to the cell membranes of intercellular canaliculi. Luminal cell membranes of secretory cells that are in continuity with intercellular canaliculi did not show ALP activity. These results suggest that ALP participates in the production of primary sweat at intercellular canaliculi. In apocrine sweat glands, basal cell membranes of secretory cells and myoepithelial cell membranes that were in apposition with each other showed ALP activity, where as no activity was seen in eccrine sweat glands. These differences in the distribution of ALP in myoepithelial cells between eccrine and apocrine sweat glands might be related to the functional differences of these sweat glands. ALP histochemistry could help to diagnose and to determine the direction of differentiation in sweat gland tumors.

The Ultrastructural Localization of Acid and Alkaline Phosphatase Via Sulfhydryl Reduction of a Nonosmiophilic Tetrazolium Salt to an Osmiophilic Formazan

1973 ◽  
Vol 31 ◽  
pp. 694-695
Author(s):  
W. Allen Shannon ◽  
Yoshinobu Hoshino ◽  
Arnold M. Seligman
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Metal Salt ◽  
Ultrastructural Localization ◽  
Enzymatic Activities ◽  
Tetrazolium Salt ◽  
Lysosomal Hydrolases ◽  
Cytochemical Localization ◽  
New Approach ◽  
Acid And Alkaline Phosphatase

The metal salt capture methods for acid phosphatase and alkaline phosphatase with numerous variations based principally on the Gomori method have been useful methods for direct cytochemical visualization of the enzymes. However, the possibilities of nonenzymatic deposition of reaction product, possible inhibition of enzymatic activities, and other suggested weaknesses of the methods which are found in the literature give reason to develop a new approach to the cytochemical localization of these enzymes. Other attempts along these lines have thus far been more or less partially successful. More recently Hanker et al. have demonstrated lysosomal hydrolases by the formation of osmium blacks based on synthetic substrates which initially deposit Hatchett's brown (cupric ferrocyanide) at the reactive sites. The synthetic substrate', 2-naph-thylthiolphosphate (NTP), developed by Seligman et al. to use in the demonstration of acid and alkaline phosphatase and subsequently used by Hanker et al.

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