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.

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.

Patterns of succinate dehydrogenase activity in a leg muscle of the domestic duck during postnatal development

1985 ◽  
Vol 63 (1) ◽  
pp. 55-57 ◽  
Author(s):  
H. J. Swatland
Keyword(s):  
Succinate Dehydrogenase ◽  
Adenosine Triphosphatase ◽  
Muscle Fibers ◽  
Histochemical Demonstration ◽  
Fiber Types ◽  
Succinate Dehydrogenase Activity ◽  
Cross Sectional ◽  
Computer Controlled ◽  
Leg Muscle ◽  
Muscovy Ducks

Transverse sections of iliotibialis cranialis from male Muscovy ducks were reacted for histochemical demonstration of myofibrillar adenosine triphosphatase (ATPase) and for succinate dehydrogenase (SDH) activities. The distribution of SDH activity within muscle fibers was measured with a microscope photometer and a computer-controlled scanning stage. From 1 to 10 weeks after hatching, the average SDH activity across muscle fiber areas decreased. All fiber types exhibited a decline of SDH activity in their central axis. However, fibers with strong ATPase and weak SDH concurrently developed stronger SDH activity in their subsarcolemmal zone. Thus, centripetal radial gradients of SDH activity within fibers became more negative as muscle fibers grew in cross-sectional area.

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.

Size and metabolic properties of fibers in rat fast-twitch muscles after hindlimb suspension

1987 ◽  
Vol 62 (6) ◽  
pp. 2348-2357 ◽  
Author(s):  
R. R. Roy ◽  
M. A. Bello ◽  
P. Bouissou ◽  
V. R. Edgerton
Keyword(s):  
Fiber Type ◽  
Female Rats ◽  
Medial Gastrocnemius ◽  
Hindlimb Suspension ◽  
Staining Reaction ◽  
Fiber Types ◽  
Image Analysis System ◽  
Cross Sectional ◽  
Glycerophosphate Dehydrogenase ◽  
Metabolic Marker

Hindlimb suspension (HS) results in whole muscle atrophic and metabolic changes that vary in magnitude in different hindlimb muscles. The present study was designed to investigate these effects in single fibers. Fiber type and size and the activities of two metabolic marker enzymes were determined in a deep (close to the bone) and a superficial (away from the bone) region of the medial gastrocnemius (MG) and the tibialis anterior (TA) of control (CON) and 28-day HS adult female rats. Fibers were classified as dark or light adenosinetriphosphatase (ATPase) based on their qualitative staining reaction for myosin ATPase following alkaline preincubation. Fiber area and succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities were determined in tissue sections by use of an image analysis system. After 28 days of HS, the mean body weights of the CON and HS were similar. MG atrophied 28%, whereas TA weight was maintained in the HS. Both dark and light ATPase fibers in the deep region of the MG had smaller cross-sectional areas following HS, with the atrophic response being approximately twice as great in the light ATPase fibers. No significant changes in fiber type composition in either muscle or in fiber sizes in the superficial region of the MG or in either region of the TA were observed. Mean SDH activities of both fiber types were significantly lower in the MG and TA following HS. In contrast, mean GPD activities were either increased or maintained in light and dark ATPase fibers of both muscles in HS. Changes in SDH and GPD activity could not be directly linked to changes in fiber cross-sectional area. In summary, these data suggest an independence of the mechanisms determining muscle fiber size and metabolic adaptations associated with HS.

Intracellular distribution of succinate dehydrogenase activity in skeletal muscle fibers of geese

1984 ◽  
Vol 62 (2) ◽  
pp. 235-240 ◽  
Author(s):  
H. J. Swatland
Keyword(s):  
Skeletal Muscle ◽  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Adenosine Triphosphatase ◽  
Muscle Fibers ◽  
Fiber Types ◽  
Succinate Dehydrogenase Activity ◽  
Individual Muscle ◽  
Concentric Zone ◽  
Pectoralis Muscles

Samples of iliotibialis anterior and pectoralis muscles were taken from five ganders (Anser domesticus). Serial transverse sections were reacted for succinate dehydrogenase (SDH) and alkali-stable adenosine triphosphatase (ATPase). The distribution of SDH activity within individual muscle fibers was measured with a scanning photometer. In many individual fibers, SDH activity was stronger in the periphery than in the axis. This gradient was steepest (−0.034 ± 0.019 absorbance units per concentric zone of 2 μm diameter measurements) in pectoralis fibers with strong SDH activity. In the pectoralis, radial gradients were correlated with fiber area so that the smallest fibers tended to have the steepest gradients of SDH activity. However, this relationship was reversed in fibers with strong ATPase and weak SDH activity in the iliotibialis anterior, and the largest fibers tended to have the steepest gradients. In all fiber types of both muscles, fibers with greater mean SDH activity tended to have steeper gradients.

scholarly journals Flexibility of an Active Center in Sodium-Plus-Potassium Adenosine Triphosphatase

1969 ◽  
Vol 54 (1) ◽  
pp. 306-326 ◽  
Author(s):  
R. L. Post ◽  
S. Kume ◽  
T. Tobin ◽  
B. Orcutt ◽  
A. K. Sen
Keyword(s):  
Active Center ◽  
Adenosine Triphosphate ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Adenosine Diphosphate ◽  
Ion Concentration ◽  
Potassium Ions ◽  
Sodium Ions ◽  
Intact Cells ◽  
Electrophoretic Patterns

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms. It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.

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