scholarly journals QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS: ADENOSINE TRIPHOSPHATASE ACTIVITY IN NORMAL AND OBESE-HYPERGLYCEMIC MICE

1964 ◽  
Vol 12 (7) ◽  
pp. 491-497 ◽  
Author(s):  
INGE-BERT TÄLJEDAL ◽  
BO HELLMAN ◽  
CLAES HELLERSTRÖM
Keyword(s):  
Enzyme Activity ◽  
Adenosine Triphosphatase ◽  
Endocrine Pancreas ◽  
Islet Cells ◽  
Adenosine Diphosphate ◽  
Histochemical Staining ◽  
Isolated Pancreatic Islets ◽  
Quantitative Studies ◽  
Substrate Cleavage ◽  
Substrate Medium

Microchemical and histochemical methods were used for the characterization, localization and assay of adenosine triphosphate (ATP) splitting enzymes in homogenates and sections of the endocrine pancreas from obese-hyperglycemic mice and their lean litter mates. The following observations were made: 1. Dephosphorylation of ATP was maximal at pH 9.1. It was strongly stimulated by magnesium ions at an optimal concentration of 1 mM. ATP cleavage was inhibited by adenosine diphosphate, sodium azide and p-chloromercuribenzoic acid. The addition of l-cysteine, sodium cyanide or sodium fluoride to the substrate medium had no effect on the enzyme activity. Substitution of ATP by equimolar amounts of other phosphate esters in the medium considerably reduced the substrate cleavage. These results are taken as evidence for the presence of sulfhydryl-dependent adenosine triphosphatase (ATPase) in the islet tissue. 2. Histochemical staining revealed a strong ATP splitting enzyme activity in the capillaries and walls of larger blood vessels throughout the pancreas; a rather weak and diffuse cytoplasmic reaction being found in the islet cells. 3. Microchemical assays revealed a lower cleavage of ATP in the islets as compared with the exocrine pancreas and the liver. The cleavage of ATP was more intense in the islets of the obese-hyperglycemic mice than in those of the lean litter mates. 4. Starvation for 7 days induced no significant changes in the enzyme activity of the endocrine pancreas.

QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS

1963 ◽  
Vol 42 (4) ◽  
pp. 615-624 ◽  
Author(s):  
Claes Hellerström ◽  
Bo Hellman
Keyword(s):  
Enzyme Activity ◽  
B Cells ◽  
Enzyme Level ◽  
Enzyme Reaction ◽  
Histochemical Staining ◽  
Pancreatic Tissue ◽  
Free Access ◽  
Isolated Pancreatic Islets ◽  
Insulin Synthesis ◽  
Dipeptidase Activity

ABSTRACT Microtitrimetric assays of dipeptidase activity were performed in isolated pancreatic islet tissue from mice. Considerable enzyme activity was found in both the endocrine and exocrine pancreas of normal mice, the enzyme level of the exocrine parenchyma being significantly higher. In obesehyperglycaemic mice with free access to food, isolated islets of Langerhans had a much higher enzyme activity than in normal animals. The increased islet dipeptidase activity in the obese-hyperglycaemic animals may, at least in part, be accounted for by their higher proportion of B cells. The intense insulin synthesis and renewal of B cells in these animals have been considered as alternative explanations. Histochemical staining for leucine aminopeptidase revealed a moderate enzyme reaction in both the endocrine and exocrine pancreatic tissue of normal and obese-hyperglycaemic mice.

HISTOCHEMICAL STUDIES ON GLUCOSE-6-PHOSPHATASE, ADENOSINE TRIPHOSPHATASE AND AMYLO PHOSPHORYLASE IN THE PANCREATIC ISLETS OF NORMAL AND OBESE-HYPERGLYCAEMIC MICE

1962 ◽  
Vol 39 (3) ◽  
pp. 474-482 ◽  
Author(s):  
Bo Hellman ◽  
Claes Hellerström
Keyword(s):  
Enzyme Activity ◽  
B Cells ◽  
Connective Tissue ◽  
Substrate Specificity ◽  
Pancreatic Islets ◽  
Islets Of Langerhans ◽  
Adenosine Triphosphatase ◽  
Islet Cells ◽  
Hydrolyzing Enzymes ◽  
Hereditary Obesity

ABSTRACT Histochemical methods for the detection of glucose-6-phosphatase (G-6-Pase), adenosine triphosphatase (ATPase) and amylo phosphorylase were applied to the islets of Langerhans in mice with hereditary obesity and hyperglycaemia (AO-mice) and in their lean litter mates (AN-mice). While the G-6-Pase activity was high in the hyperactive B cells of the AO-mice, it was only moderate in the AN-mice. In both types of mice ATP-hydrolyzing enzymes were observed in the islet capillaries and periinsular connective tissue, while the reaction in the islet cells was negative. From a study of the substrate specificity it was found that the enzyme activity was not dependent on specific ATPase, but probably mainly on less specific polyphosphatases. The reaction for amylo phosphorylase was negative in the islets of both the AN- and the AO-mice. The high G-6-Pase activity in the islets of the AO-mice is discussed in the light of the hypothesis that the activity of this enzyme represents an essential controlling factor in the insulin output of the B-cells.

scholarly journals AN IMPROVED METHOD OF FIXATION FOR FORMALIN-SENSITIVE ENZYMES WITH SPECIAL REFERENCE TO MYOSIN ADENOSINE TRIPHOSPHATASE

1966 ◽  
Vol 14 (8) ◽  
pp. 577-581 ◽  
Author(s):  
MASANDO HAYASHI ◽  
DAVID G. FREIMAN
Keyword(s):  
Succinate Dehydrogenase ◽  
Adenosine Triphosphatase ◽  
Adenosine Diphosphate ◽  
Histochemical Staining ◽  
Thiamine Pyrophosphate ◽  
Staining Reaction ◽  
Appreciable Effect ◽  
Improved Method ◽  
Glycerophosphate Dehydrogenase ◽  
Leg Muscle

Studies have been made on the effect of fixatives containing substrate and other additives on the histochemical staining reaction for myosin adenosine triphosphatase activity in the leg muscle, and α-glycerophosphate and succinate dehydrogenase activities in the kidney of the rat. Myosin adenosine triphosphatase was well preserved in the presence of adenosine triphosphate in fixative prepared from methanol-free formaldehyde and buffered to pH 7.2 with cacodylate. Addition of sucrose was found to increase the enzyme preservation. Similar protection of the enzyme activity was afforded by other polyphosphates, particularly sodium pyrophosphate and, to less degree, thiamine pyrophosphate, adenosine diphosphate and uridine triphosphate. α-Glycerophosphate dehydrogenase was also preserved to greater degree by a similar fixative containing α-glycerophosphate than by substrate-free fixative. Succinate dehydrogenase was not significantly preserved in succinate-containing fixative. On the other hand, sucrose in the fixative increased the preservation of succinate dehydrogenase but had no appreciable effect on α-glycerophosphate dehydrogenase.

QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS: ENZYMIC HYDROLYSIS OF NUCLEOSIDE DIPHOSPHATES AND p-NITROPHENYL PHOSPHATE IN NORMAL AND CORTISONE-TREATED RATS

1966 ◽  
Vol 36 (2) ◽  
pp. 115-NP ◽  
Author(s):  
I.-B. TÄLJEDAL ◽  
B. HELLMAN ◽  
C. HELLERSTRÖM
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Sodium Fluoride ◽  
Acid Phosphatase Activity ◽  
Staining Intensity ◽  
Histochemical Staining ◽  
Enzymic Hydrolysis ◽  
Isolated Pancreatic Islets ◽  
Hydrolysis Of

SUMMARY Chemical micromethods and histochemical staining were employed for studies of the enzymic hydrolysis of inosine diphosphate (IDP) and adenosine diphosphate (ADP) and the non-specific acid phosphatase activity of the endocrine pancreas from normal and cortisone-treated rats. The following observations were made: 1. Enzymic dephosphorylation of IDP and ADP was maximal at about pH 8·0. Magnesium and manganese ions enhanced the phosphate liberation, the hydrolysis of ADP being more activated than that of IDP. A marked inhibition of enzyme activity towards either substrate was produced by sodium fluoride, sodium cyanide and ethylene-diaminotetraacetate. Acid phosphatase activity was maximal at about pH 5·5, a tendency for a second activity optimum was noted at about pH 4·0. Acid phosphatase activity was markedly inhibited by sodium fluoride, tartaric acid and formaldehyde. 2. Histochemical staining revealed marked enzyme activity towards IDP and ADP in the capillaries and walls of the large blood vessels throughout the pancreas, whereas the islet cells displayed a moderate reaction. The staining intensity was the same with IDP as with ADP. 3. Cortisone administration reduced the rate of cleavage of both IDP and ADP in both the endocrine and the exocrine pancreas, but the enzymic splitting of these substrates remained unchanged in the liver. Acid phosphatase activity was not influenced in any of these tissues by the steroid treatment.

scholarly journals Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

1975 ◽  
Vol 23 (5) ◽  
pp. 342-347 ◽  
Author(s):  
A Linde ◽  
B C Magnusson
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Adenosine Triphosphatase ◽  
Ruthenium Red ◽  
Inorganic Pyrophosphatase ◽  
Alkaline Ph ◽  
Hard Tissue ◽  
Assay Condition ◽  
Phosphatase Inhibitors ◽  
Inhibition Studies

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF Mg++-DEPENDENT DINITROPHENOL-STIMULATED ADENOSINE TRIPHOSPHATASE IN HUMAN BLOOD PLATELETS

1967 ◽  
Vol 15 (5) ◽  
pp. 267-272 ◽  
Author(s):  
VICTOR G. VETHAMANY ◽  
SYDNEY S. LAZARUS
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Blood ◽  
Adenosine Triphosphatase ◽  
Blood Platelets ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Structural Localization ◽  
Adenosine Triphosphatase Activity ◽  
Human Blood Platelets

Fine structural localization of adenosine triphosphatase activity was studied in human platelets briefly fixed in cold formol calcium and then incubated in lead medium with added dinitrophenol. Under these conditions, the Mg++-dependent dinitrophenol-stimulated adenosine triphosphatase of platelet mitochondria was demonstrated, but neither granules nor plasma membrane showed enzyme activity.

scholarly journals QUANTITATIVE AND HISTOCHEMICAL STUDIES ON THE DESORPTION AND READSORPTION OF ALDOLASE IN CROSS-STRIATED MUSCLE

1969 ◽  
Vol 17 (5) ◽  
pp. 314-320 ◽  
Author(s):  
H. ARNOLD ◽  
J. NOLTE ◽  
D. PETTE
Keyword(s):  
Enzyme Activity ◽  
Actin Filaments ◽  
Phosphate Buffer ◽  
Striated Muscle ◽  
Psoas Muscle ◽  
Histochemical Staining ◽  
Intracellular Location ◽  
Successive Extraction ◽  
Complete Extraction

Complete extraction of aldolase from minced rabbit psoas muscle was achieved by successive extraction steps in 0.1 M phosphate buffer. Aldolase was then readsorbed quantitatively to the depleted myofibrils. Extraction, readsorption and a final redsorption of the enzyme were followed quantitatively by enzyme activity determinations and qualitatively by histochemical staining of aldolase. The intracellular location of the readsorbed enzyme was found to be identical with that of aldolase in native muscle. In both cases, aldolase was localized within the isotropic bands. These results as well as the previously demonstrated binding of the enzyme to F-actin suggest that aldolase is located within the interfilamentary sarcoplasm of the isotropic bands and is probably also bound in vivo to the actin filaments.

scholarly journals ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

1963 ◽  
Vol 19 (2) ◽  
pp. 337-347 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Enzyme Activity ◽  
Hela Cells ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Simplex Virus ◽  
Vacuolar Membranes

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.

Fine-Structural Localization of Adenosine Triphosphatase in the Rectum of Calliphora

1968 ◽  
Vol 3 (1) ◽  
pp. 17-32
Author(s):  
M. J. BERRIDGE ◽  
B. L. GUPTA
Keyword(s):  
Plasma Membrane ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Fluid Transport ◽  
Microsomal Fraction ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Osmotic Gradient ◽  
Ph Optimum ◽  
Structural Localization

Adenosine triphosphatase (ATPase) activity in the rectal papillae of Calliphora has been studied by biochemical and histochemical techniques. The microsomal fraction contained a Mg2+-activated ATPase with a pH optimum of 8.0. The enzyme was not stimulated by the addition of Na+ plus K+ and was insensitive to ouabain. Histochemical studies using modifications of the Wachstein-Meisel method showed that at pH 7.2 this Mg2+-activated ATPase was specifically localized on the intracellular surface of the lateral plasma membranes. A similar though less intense reaction was obtained with adenosine diphosphate and inosine triphosphate, but not with guanosine triphosphate, uridine triphosphate or β-glycerophosphate as substrates. At an acid pH (6.6-6.8), very little reaction occurred on the lateral plasma membrane but some reaction product was present in mitochondria and nuclei. Very little enzyme activity was found in the flattened rectal epithelium. These results are discussed in relation to the available data on transport ATPases and on the structural basis of fluid transport by rectal papillae. It is proposed that the ATPase localized on the stacks of lateral plasma membrane may be involved with ion secretion into the intercellular spaces to create the osmotic gradient necessary to extract water from the lumen.

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