scholarly journals Biochemical and cytochemical localization of inosine-5′-diphosphatase in rat liver microsomal fractions

1984 ◽  
Vol 218 (2) ◽  
pp. 501-510 ◽  
Author(s):  
J S Little
Keyword(s):  
Electron Microscope ◽  
Rat Liver ◽  
Incubation Medium ◽  
Microsomal Membrane ◽  
Enzymic Hydrolysis ◽  
Major Fraction ◽  
Cytochemical Localization ◽  
Hydrolysis Of ◽  
Cytoplasmic Side

A procedure has been developed for the biochemical and cytochemical localization of inosine-5′-diphosphatase (IDPase) in rat liver microsomal fractions. The concentration of Pb2+ ions used in the cytochemical incubation medium inhibited IDPase by 32%. IDP formed during the incubation was not hydrolysed further to inosine, and no significant non-enzymic hydrolysis of substrate occurred. When microsomal fractions were incubated cytochemically for IDPase and separated from unchanged substrate under conditions such that the microsomal membrane was not permeable to EDTA, approx. 80% of the reaction product was solubilized by EDTA. In the electron microscope, lead precipitate was observed on the cytoplasmic side (outside) of a major fraction of the vesicles and on the cisternal side (inside) of others. After extraction with EDTA, the lead precipitate was observed only on the cisternal side. Since it was shown that phosphate is trapped on the same side of the membrane as it is released, it is concluded that IDPase can release phosphate on either the cisternal or the cytoplasmic side of the microsomal membrane.

Ultrastructural Localization of 5'-Nucleotide Phosphodiesterase in Rat Liver and Hepatoma

1973 ◽  
Vol 31 ◽  
pp. 330-331
Author(s):  
K. C. Tsou ◽  
P. D. Gupta
Keyword(s):  
Electron Microscope ◽  
Enzymatic Hydrolysis ◽  
Rat Liver ◽  
Present Report ◽  
Ultrastructural Localization ◽  
Lead Phosphate ◽  
Hydrolytic Product ◽  
Cytochemical Method ◽  
Hydrolysis Of

The difficulty in developing a 5'-nucleotide phosphodiesterase cytochemical method suitable for electron microscope is well-known. Unlike ATPase or 5′-nucleotidase, where the insoluble lead phosphate can be used successfully to localize the hydrolytic product, both products from enzymatic hydrolysis of a 5′-nucleotide phosphodiester are soluble. Earlier, an indigogenic substrate, 5′-(5-iodo-3-indolyl)-thymidine phosphodiester was used to yield an insoluble 5,5′-di-iodoindigo. This method was ultimately improved with the use of a mixture of 5′-(5-iodo-3-indolyl)-5-fluorodeoxyuridine (5′-IIp-FUdR), and 5′-(5-nitro-3-indolyl)-5-fluorodeoxyuridine (5′- N02lp-FUdR), which produced an insoluble electron-dense indigo complex. The present report demonstrates its usefulness for the ultrastructural localization of this enzyme in rat liver and hepatoma.

scholarly journals Enzymic hydrolysis of sphingomyelin by rat liver

1966 ◽  
Vol 98 (3) ◽  
pp. 763-769 ◽  
Author(s):  
M Heller ◽  
B Shapiro
Keyword(s):  
Rat Liver ◽  
Enzymic Hydrolysis ◽  
Hydrolysis Of

scholarly journals Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. II. Cytochemical localization within Golgi and ER membranes.

1976 ◽  
Vol 70 (3) ◽  
pp. 671-684 ◽  
Author(s):  
H Cheng ◽  
M G Farquhar
Keyword(s):  
Adenylate Cyclase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Smooth Endoplasmic Reticulum ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lead Phosphate ◽  
Cytochemical Localization ◽  
Cell Fractions ◽  
Hydrolysis Of

The presence of adenylate cyclase (AC) in liver Golgi and microsomal fractions from ethanol-treated rats was tested cytochemically using 5'-adenylyl imidodiphosphate (AMP-PNP) lead phosphate method. Parallel biochemical assays showed that rat liver Golgi AC was only partially inhibited by lead: in the presence of 1 mM Pb++ 80% of the enzyme was preserved, while when 2 mM Pb++ was used 25% remained. No cAMP was formed when the AMP-PNP medium was incubated in the presence of 1 or 2 mM Pb++ but in the absence of cell fractions, indicating that at these concentrations Pb++ does not cause the nonenzymatic hydrolysis of AMP-PNP. Therefore, the reaction product observed by cytochemical localization is not due to the nonenzymatic hydrolysis of AMP-PNP by Pb++. In Golgi subfractions, lead phosphate reaction product was widely distributed among Golgi elements: it was seen in association with the majority of the very low density lipoprotein-filled secretory droplets which predominated in the two lightest Golgi fractions (GF1 and GF2) as well as within the majority of the cisternae found in the heaviest Golgi fraction (GF3). In the latter, reaction product was heaviest along the dilated peripheral rims of the cisternae. In all cases, the reaction product was localized to the outside or cytoplasmic face of the Golgi membranes. When microsomes were incubated cytochemically for AC, deposits were found on the cytoplasmic surface of smooth endoplasmic reticulum (ER) membranes, but none were observed on rough ER membranes. The results confirm the biochemical data reported previously indicating the presence of AC in Golgi and smooth microsomal fractions from rat liver and further demonstrate that the activity is indeed indigenous to Golgi elements and not due to plasma membrane contaminants. They also indicate that AC is widely distributed among Golgi and smooth ER elements. Thus, AC is not restricted in its distribution to plasma membranes as usually assumed.

scholarly journals Mechanism of hydroxylation at C-22 during the biosynthesis of ecdysteroids in the locust Schistocerca gregaria

1982 ◽  
Vol 208 (3) ◽  
pp. 857-864 ◽  
Author(s):  
D R Greenwood ◽  
H H Rees
Keyword(s):  
Rat Liver ◽  
Adult Female ◽  
Schistocerca Gregaria ◽  
Mevalonic Acid ◽  
Enzymic Hydrolysis ◽  
Hydrogen Atoms ◽  
Hydrolysis Of

1. The fates of the 22-pro-R and 22-pro-S hydrogen atoms of cholesterol during the biosynthesis of ecdysteroids in the ovaries of Schistocerca gregaria were investigated. 2. Two stereospecifically labelled cholesterol species, obtained by incubating 3R,2R- and 3R,2S-[2-14C, 2-3H]mevalonic acid with rat liver preparations, were administered, in turn, to maturing adult female locusts and the radiolabelled ecdysteroid conjugates isolated from the eggs. Enzymic hydrolysis of the conjugates yielded free ecdysteroids, from which ecdysone was purified. 3. Derivative formation and oxidation at C-22 of both ecdysone samples indicated that the 22-pro-R and 22-pro-S hydrogen atoms of cholesterol were stereospecifically eliminated and retained respectively during ecdysteroid formation. This indicates that C-22 hydroxylation in ecdysone biosynthesis is direct and occurs with retention of configuration.

A Dye Phosphate for the Histo- and Cytochemical Demonstration of Alkaline Phosphatase, with some Observations on the Differential Behaviour of Nuclear and Extranuclear Enzymes

1949 ◽  
Vol s3-90 (9) ◽  
pp. 57-66
Author(s):  
A. LOVELESS ◽  
J. F. DANIELLI
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Histochemical Demonstration ◽  
Optimal Conditions ◽  
Enzymic Hydrolysis ◽  
Cytochemical Localization ◽  
Cytochemical Demonstration ◽  
End Products ◽  
Differential Behaviour ◽  
Hydrolysis Of

1. A new method is described for the cyto- and histochemical demonstration of alkaline phosphatase (monoesterase), a synthetic substrate p-nitrobenzene-azo-4α-naphthol-phosphate being employed as the sodium salt. Two methods for preparing a solution of this substance are described, and optimal conditions for enzymic hydrolysis of the ester worked out by the use of kidney sections. 2. Experiments are also described in which the substrate has been used to demonstrate phosphatase activity in calcifying tissues of dogfish and rat. 3. The results of the experiments with rat kidney indicate that traces of end-products are necessary before the enzyme can attack this substrate. 4. When results obtained with the new substrate are compared with those obtained with β-glycerophosphate, it is found that some sites display more activity towards one substrate than to the other. 5. The cytochemical localization of phosphatase with the new substrate is not as precise as with β-glycerophosphate.

Initial Polymerization Sites Indicated During Mitochondrial Oxidation of Diaminobenzidine after Toluene Treatment

1977 ◽  
Vol 35 ◽  
pp. 408-409
Author(s):  
W. A. Shannon ◽  
M. A. Matlib
Keyword(s):  
Membrane Permeability ◽  
Cytochrome Oxidase ◽  
Rat Liver ◽  
Precise Definition ◽  
Cytochemical Localization ◽  
Oxidative Reaction ◽  
Mitochondrial Oxidation ◽  
Definition Of ◽  
Exogenous Substrates

Numerous studies have dealt with the cytochemical localization of cytochrome oxidase via cytochrome c. More recent studies have dealt with indicating initial foci of this reaction by altering incubation pH (1) or postosmication procedure (2,3). The following study is an attempt to locate such foci by altering membrane permeability. It is thought that such alterations within the limits of maintaining morphological integrity of the membranes will ease the entry of exogenous substrates resulting in a much quicker oxidation and subsequently a more precise definition of the oxidative reaction.The diaminobenzidine (DAB) method of Seligman et al. (4) was used. Minced pieces of rat liver were incubated for 1 hr following toluene treatment (5,6). Experimental variations consisted of incubating fixed or unfixed tissues treated with toluene and unfixed tissues treated with toluene and subsequently fixed.

scholarly journals Hydrolysis of Retinol Palmitate by Rat Liver

1966 ◽  
Vol 241 (1) ◽  
pp. 57-64 ◽  
Author(s):  
S. Mahadevan ◽  
N.I. Ayyoub ◽  
O.A. Roels
Keyword(s):  
Rat Liver ◽  
Hydrolysis Of

Variation in Quantity of Methanol Recovered from Raisins

1963 ◽  
Vol 46 (2) ◽  
pp. 341-343
Author(s):  
M Alice Brown ◽  
James R Woodward ◽  
Floyd DeEds
Keyword(s):  
Methyl Ester ◽  
Esterase Activity ◽  
Enzymic Hydrolysis ◽  
Methanol Content ◽  
Naturally Occurring ◽  
Pectin Esterase ◽  
Hydrolysis Of

Abstract The amount of naturally occurring methanol in fruit must be known so that the quantity left as fumigation residue can be determined. In a study of methanol content of raisins, which had given inconsistent results, the raisins were subjected to different conditions of treatment immediately prior to methanol determination. Conditions that favored pectin esterase activity gave higher values for methanol content than conditions known to inactivate enzymes. Evidence was also obtained that both chemical and enzymic hydrolysis of methyl ester groups of pectic materials occur during analysis.

scholarly journals Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels.

1975 ◽  
Vol 64 (3) ◽  
pp. 586-607 ◽  
Author(s):  
N Simionescu ◽  
M Siminoescu ◽  
G E Palade
Keyword(s):  
Plasma Proteins ◽  
Intercellular Junctions ◽  
Rat Diaphragm ◽  
Enzymic Hydrolysis ◽  
Graphic Analysis ◽  
Heme Peptides ◽  
Future Work ◽  
Hydrolysis Of ◽  
Muscle Capillaries

Two heme-peptides (HP) of about 20-A diameter (heme-undecapeptide [H11P], mol wt approximately 1900 and heme-octapeptide [H8P], mol wt approximately 1550), obtained by enzymic hydrolysis of cytochrome c, were sued as probe molecules in muscle capillaries (rat diaphragm). They were localized in situ by a perixidase reaction, enhanced by the addition of imidazole to the incubation medium. Chromatography of plasma samples showed that HPs circulate predominantly as monomers for the duration of the experiments and are bound by aldehyde fixatives to plasma proteins to the extent of approximately 50% (H8P) to approximately 95% (H11P). Both tracers cross the endothelium primarily via plasmalemmal vesicles which become progressively labeled (by reaction product) from the blood front to the tissue front of the endothelium, in three successive resolvable phases. By the end of each phase the extent of labeling reaches greater than 90% of the corresponding vesicle population. Labeled vesicles appear as either isolated units or chains which form patent channels across the endothelium. The patency of these channels was checked by specimen tilting and graphic analysis of their images. No evidence was found for early or preferential marking of the intercellular junctions and spaces by reaction product. It is concluded that the channels are the most likely candidate for structural equivalents of the small pores of the capillary wall since they are continuous, water-filled passages, and are provided with one or more strictures of less than 100 A. Their frequency remains to be established by future work.

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