Showing papers on "Alkaline phosphatase published in 1968"

Rat intestinal microvillus membranes. Purification and biochemical characterization

Abstract: 1. A technique is described for the removal of subcellular contaminants from intact rat intestinal brush borders, and for the subsequent separation of a microvillus membrane fraction from a fibrillar residue. 2. Increments in invertase activity, microscopic homogeneity and low nucleic acid content indicate that the microvillus plasma membrane has been extensively purified. Multiple membrane preparations have been shown to be highly reproducible with respect to their invertase specific activity, cholesterol content and phospholipid content. Alkaline phosphatase, leucine aminopeptidase, Mg2+- and Ca2+-dependent adenosine triphosphatase and seven separate disaccharidases were shown to be predominantly confined to the membrane fraction. 3. The fibrillar fraction has been shown to contain approximately 30% of the total protein of purified brush borders, plus most of the residual nucleic acid contaminant. No evidence was found for the localization of any specific enzyme in this fraction.

A Serum Alkaline Phosphatase Isoenzyme of Human Neoplastic Cell Origin

Abstract: Summary In the course of a study of serum phosphatase isoenzymes in cancer patients, a patient with bronchogenic carcinoma was discovered, whose serum contained an alkaline phosphatase which was indistinguishable from placental alkaline phosphatase. The same enzyme was demonstrated in high concentration in the neoplasm and its metastases. This single case demonstrates the possibility that elevated serum alkaline phosphatase in patients with metastatic cancer can be of neoplastic rather than of hepatic and osseous origin.

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES: II. Cytochemistry and Electron Microscopy of Bone Marrow Cells

Abstract: In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 59-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product.

On the mechanisms of bone resorption. The action of parathyroid hormone on the excretion and synthesis of lysosomal enzymes and on the extracellular release of acid by bone cells.

Abstract: Bone resorption, characterized by the solubilization of both the mineral and the organic components of the osseous matrix, was obtained in tissue culture under the action of parathyroid hormone (PTH). It was accompanied by the excretion of six lysosomal acid hydrolases, which was in good correlation with the progress of the resorption evaluated by the release of phosphate, calcium 45 or hydroxyproline from the explants; there was no increased excretion of two nonlysosomal enzymes, alkaline phosphatase, and catalase. Balance studies and experiments with inhibitors of protein synthesis indicated that the intracellular stores of the acid hydrolases excreted were maintained by new synthesis. The release was not due to a direct disruption of the lysosomal membrane by PTH; it is presumed to result from an exocytosis of the whole lysosomal content and to involve mechanisms similar to those controlling the secretion of this content into digestive vacuoles. The resorbing explants acidified their culture fluids at a faster rate and released more lactate and citrate than the controls; this release was in good correlation, in the PTH-treated cultures, with the resorption of the bone mineral, but the amount of citrate released was considerably smaller than that of lactate. The acid released could account for the resorption of the mineral. It is proposed, as a working hypothesis, that the acid hydrolases of the lysosomes are active in the resorption of the organic matrix of bone and that acid, originating possibly from the stimulation of glycolysis, cares for the concomitant solubilization of bone mineral while also favoring the hydrolytic action of the lysosomal enzymes.

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : I. Histochemical Staining of Bone Marrow Smears

Abstract: 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, β-galactosidase, β-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.

Immunology and biochemistry of Regan isoenzyme of alkaline phosphatase in human cancer.

Abstract: Derepression of the genome of the cancer cell may explain why certain cancer patients exhibit an isoenzyme of alkaline phosphatase biochemically and immunologically indistinguishable from human placental alkaline phosphatase.

Influence of pyrophosphate on the transformation of amorphous to crystalline calcium phosphate

Abstract: The transformation of amorphous calcium phosphate into its crystalline form has been studiedin vitro under various conditions. The transformation was followed by changes in the pH and in the calcium and phosphate content of the solution and by changes in the Ca/P ratio and x-ray diffraction patterns of the solid phase. It was found that inorganic pyrophosphate markedly increased the time required for the transformation under the various conditions used. The addition of intestinal alkaline phosphatase abolished this retarding effect of pyrophosphate on the transformation. It is proposed that pyrophosphate may be one of the factors that allows part of the bone mineral to persist in a non-crystalline state. The alkaline phosphatase of bone, by virtue of its pyrophosphatase activity, might be able to accelerate the transformation processin vivo.

The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli.

Abstract: 1. The steady-state rate of hydrolysis of 2,4-dinitrophenyl phosphate catalysed by Escherichia coli phosphatase is identical with that of 4-nitrophenyl phosphate over the pH range 5.5-8.5. 2. The increase in the rate of the enzyme-catalysed decomposition of nitrophenyl phosphates in the presence of tris at pH8.1 and 5.9 is consistent with the hypothesis that tris increases the rate of decomposition of a phosphoryl-enzyme intermediate. At pH8.1 the rate of decomposition of the phosphoryl-enzyme is approximately twice as fast as the rate of its formation, whereas at pH5.9 the rate of formation of the phosphoryl-enzyme is considerably faster than its decomposition. 3. Pre-steady-state measurements of the initial transient of the liberation of 2,4-dinitrophenol during the reaction of the enzyme with 2,4-dinitrophenyl phosphate confirmed the above pH-dependence of the ratio of the rates of phosphorylation and dephosphorylation of the enzyme. At optimum pH (above pH8), when the phosphorylation of the enzyme by the substrate is rate-determining, this step must be controlled by a rearrangement of the enzyme or enzyme-substrate complex.

Leukocyte alkaline phosphatase cytochemistry: applications and methods*

Abstract: Introduction Almost 20 years ago 0. P. Jones’ in a special issue of Blood stated, “. . . the use of dry fixed smears is not necessarily limited to the field of descriptive or morphologic hematology but, with the proper selection of experimental material, may be utilized to good advantage in determining cell potentialities.” In the same publication Dameshek wrote, “. . . morphology when appropriately interpreted, can be both physiology and chemistry. . . far from being dead, morphologic hematology is undoubtedly in for a renaissance period, a period in which it is hoped that appropriate staining techniques will point directly to chemical and physiopathologic alteration^.\"^ That such an era has indeed emerged is evident from the titles of the papers presented in this monograph and is well exemplified by the cytochemical demonstration and assessment of leukocyte alkaline phosphatase activity (LAPA). The demonstration of enzyme activity in situ is dependent upon fixation methods that will preserve cytological detail with minimal inhibition of enzyme activity and upon an enzyme-substrate reaction product which is either insoluble in its immediate environment or which can combine to form an insoluble product with other substances already present in, or to be added to, the incubation mixture. Preferably, this product should be colored so as to be visible under ordinary transmitted light, or a colorless product may be reacted on by other reagents SO as to produce a final colored precipitate that is localized at the theoretical sites of enzyme activity. There are important advantages to be gained in the study of blood, which’are denied when working with solid tissues. Blood smears, monocellular in thickness, permit the briefest of fixation and staining times and require neither embedding nor mounting. Furthermore, cellular units are intact; the cell membrane is not cut as is unavoidable in tissue sections. The intracellular detail that can be discerned in properly prepared blood smears is generally superior to that seen on sectioned tissues. Above all, blood is a unique tissue. It is readily biopsied and a minute sample is not only representative of the circulating blood-cell population but may also reflect the physiological state of the whole organism. Blood cells have been categorized according to their morphology and on the basis of their tinctorial staining characteristics (Giemsa-Wright’s stain). Thus, mature neutrophilic granulocytes have been thought of as members of a homogeneous group of cells with similar function and similar capabilities. Cytochemical techniques readily destroy such concepts. This is well exemplified by staining for LAPA, which reveals that two adjacent neutrophils, lying within a single microscopic field, appearing almost identical by conventional staining techniques, may differ markedly in alkaline phosphatase activity (FIGURE 1). Whether these

Fine structural localization of acid and alkaline phosphatase activities in the absorbing cells of the duodenum of rodents

Abstract: The morphology of the absorbing cells of the duodenal villi in the mouse, the rat, the hamster and the guinea-pig is described. The polymorphism of the dense bodies is pointed out. The fine localization of acid and alkaline phosphatase is investigated and compared. In all the species, acid phosphatase activity is observed in the dense bodies, Golgi vesicles and rare smooth endoplasmic profiles. Alkaline phosphatase is localized on the microvilli, Golgi apparatus, some smooth endoplasmic cisternae and numerous dense bodies. The presence of an alkaline phosphatase reaction in the dense bodies, probably lysosomes, of the absorbing cells is discussed. It is assumed that this enzyme follows a catabolic pathway and is finally degraded in the lysosomes.

Locus of the Lethal Event in the Serum Bactericidal Reaction

Abstract: Hypertonic sucrose inhibited the bactericidal activity of lysozyme-free serum against a rough strain of Escherichia coli. The duration of the inhibition correlated with the duration of plasmolysis caused by the sucrose. Although the lethal action of the serum was delayed, the prompt release of alkaline phosphatase by the cells suggested that nonlethal damage to the cell wall had taken place under these conditions. In contrast, the crypticity of the cells for β-galactosidase did not deteriorate until the viability of the bacteria began to decrease. It is concluded that the primary site of action of serum is at the bacterial cell wall; however, in the absence of lysozyme, the lethal event was subsequent damage to the bacterial cell membrane.

Inhibition of the Nucleotidase Effect of Alkaline Phosphatase by β-Glycerophosphate

Abstract: THE co-existence of more than one enzyme which hydrolyses the same substrate creates considerable difficulties when it is important to distinguish between them. Many attempts have been made to assess the hydrolysis of adenosine 5′ monophosphate (5′ AMP) by non-specific alkaline phosphatase (APase, EC 3.1.3.1) when measuring the activity of 5′ nucleotidase (EC 3.1.3.5). These include the use of 5′ AMP and β-glycerophosphate in independent assays for true nucleotidase and nucleotidase+APase1; inhibition of APase by EDTA2 and by L-histidine3; and inhibition of nucleotidase by Ni++ (ref. 4). These techniques measure the phosphorus liberated by enzyme activity, at least two assays each in different sets of conditions being required. To the imprecision resulting from the summation of random errors in each assay is added uncertainty regarding the validity of the assumptions adopted, as the extent to which Ni++ partially inhibits APase is in dispute5,6, and the conditions for EDTA inhibition are difficult to define7.

Alkaline Phosphatase Subunits and Their Dimerization In Vivo

Abstract: A pool of alkaline phosphatase subunits has been found in cells of Escherichia coli which are actively synthesizing the enzyme. The radioactive subunits from pulse-labeled cells were specifically recognized by their capacity to produce, upon incubation with Zn++ and nonradioactive monomers, radioactive dimers with the characteristics of alkaline phosphatase. The pool of subunits was larger (10 times or more) than the amount expected to be bound to ribosomes and was bound to a rapidly sedimentable fraction from which 60% was released by ribonuclease. In a culture pulse-labeled for one-third (8 sec) of the enzyme synthetic time, the pool of radioactive monomers was 81% of the radioactive enzyme and was totally (98%) in the endoplasm. The size of the pool was increased by decreasing the dimerization rate without affecting protein synthesis. This was achieved by decreasing Zn++ in the growth medium. It was found that the cells contained a full complement of monomers, although the level of active enzyme was low. A process subsequent to the release of the monomers from the ribosomes was found to be limiting the formation of the finished enzyme. This process affects the level of the pool of monomers independently from their synthesis.