LOCALIZATION AND CHARACTERIZATION OF CHOLINESTERASE IN SUBCELLULAR FRACTIONS OF RAT BRAIN AND BEEF PITUITARY

1961 ◽  
Vol 39 (9) ◽  
pp. 1335-1345 ◽  
Author(s):  
Surendra S. Parmar ◽  
Morley C. Sutter ◽  
Mark Nickerson
Keyword(s):  
Rat Brain ◽  
Cholinesterase Activity ◽  
Succinic Dehydrogenase ◽  
Anterior Lobe ◽  
Subcellular Fractions ◽  
Posterior Pituitary ◽  
Low Concentrations ◽  
Pituitary Glands ◽  
Hydrolysis Of

Fresh rat brains and fresh anterior and posterior pituitary glands of beef were separated by differential centrifugation into subcellular fractions, characterized on the basis of sedimentation and succinic dehydrogenase activity. Cholinesterase activity was measured by both manometric and colorimetric methods, the results of which were comparable. Cholinesterase activity of rat brain was found mainly in the microsome and supernatant fractions. It was quite uniformly distributed in all subcellular fractions of both anterior and posterior pituitary. Comparisons of the relative rates of hydrolysis of acetylthiocholine and butyrylthiocholine, and of inhibition by eserine, indicated that brain contains a much higher percentage of acetylcholinesterase than do both lobes of the pituitary, which contain relatively low concentrations of the specific enzyme. Total cholinesterase activity and its sensitivity to inhibition by eserine in the posterior pituitary were found to be midway between those of the anterior lobe and of the brain, from which the posterior pituitary was derived during embryological development.

scholarly journals Phenethylamines in brain and liver of rats with experimentally induced phenylketonuria-like characteristics

1973 ◽  
Vol 132 (1) ◽  
pp. 95-100 ◽  
Author(s):  
David J. Edwards ◽  
Karl Blau
Keyword(s):  
Monoamine Oxidase ◽  
Rat Brain ◽  
Phenylalanine Hydroxylase ◽  
Brain Regions ◽  
Subcellular Fractions ◽  
Brain Cells ◽  
Blood Phenylalanine ◽  
Low Concentrations ◽  
Experimentally Induced

1. Phenethylamines were extracted from brain and liver of rats with phenylketonuria-like characteristics produced in vivo by inhibition of phenylalanine hydroxylase (EC 1.14.3.1) with p-chlorophenylalanine, with or without phenylalanine administration. To protect amines against oxidation by monoamine oxidase, pargyline was also administered. 2. β-Phenethylamine was the major compound found in brain and liver. β-Phenethanolamine and octopamine were also present, in lesser amounts, and the concentrations of these three amines paralleled blood phenylalanine concentrations. By comparison, tissues from control animals had only very low concentrations of these amines. 3. Small amounts of normetadrenaline, m-tyramine and 3-methoxytyramine were also found. 4. The inhibitors used, p-chlorophenylalanine and pargyline, gave rise to p-chlorophenethylamine and benzylamine respectively, the first via decarboxylation, the second probably by breakdown during extraction. 5. Distribution of phenethylamines in different brain regions and in subcellular fractions of rat brain cells was also investigated. The content of phenethylamine was highest in the striatum. 6. These findings are discussed in the light of changes occurring in human patients with uncontrolled phenylketonuria.

scholarly journals Cell Fractionation of Anterior Pituitary Glands from Beef and Pig

1959 ◽  
Vol 5 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Frank S. Labella ◽  
J. H. U. Brown
Keyword(s):  
Alkaline Phosphatase ◽  
Anterior Pituitary ◽  
Secretory Granules ◽  
Succinic Dehydrogenase ◽  
Subcellular Fractions ◽  
Acid Proteinase ◽  
Cell Fractionation ◽  
Ph Optimum ◽  
Pituitary Glands ◽  
The Individual

Fresh anterior pituitary glands from beef and pig were separated by differential centrifugation into subcellular fractions. Nuclei and debris were obtained at 700 g for 15 minutes, secretory granules at 7000 g for 20 minutes, mitochondria at 34,000 g for 15 minutes, and microsomes at 78,000 g for 3 hours. Electron micrographs were taken of the individual fractions. Each fraction was analyzed for nitrogen, pentosenucleic acid (PNA), and phospholipide. Beef and pig anterior lobes were quite similar in their intracellular composition as seen in the subcellular fractions. Succinic dehydrogenase was localized in mitochondria, while alkaline phosphatase was concentrated in the microsomes. A proteinase with pH optimum at 8.2 was exclusively localized. in microsomal and supernatant fractions. Acid phosphatase, acid ribonuclease, and acid proteinase were distributed among the subcellular fractions in another pattern, indicating the presence of a particle type distinct from mitochondria and microsomes. The distribution of cytoplasmic PNA paralleled that of alkaline phosphatase.

Characterization of Canine Hepatic and Renal Esterases

1973 ◽  
Vol 51 (5) ◽  
pp. 506-513 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Cholinesterase Activity ◽  
Kinetic Characteristics ◽  
Migration Patterns ◽  
Tissue Extracts ◽  
Liver And Kidney ◽  
Inhibition Studies ◽  
Hydrolysis Of ◽  
Naphthyl Acetate ◽  
Substrate Hydrolysis

The esterases of canine liver and kidney were separated electrophoretically into nine bands with identical migration patterns in both tissues. An additional pair of rapidly migrating anodic bands were observed in hepatic extracts. Based on substrate specificity, the predominant tissue esterases were identified as nonspecific carboxylesterases (aliesterases). No cholinesterase activity was detected in the tissue extracts. Kinetic characteristics determined for the hepatic and renal esterases included (1) optimal pH; (2) Km values for esters of α-naphthyl and p-nitrophenol; (3) average rates of hydrolysis of α-naphthyl acetate and p-nitrophenyl acetate by the tissue extracts. Inhibition studies revealed the presence of two types of esterase activity in each tissue: one type being sensitive to organophosphorus esters, the second being resistant. A study of preferential substrate hydrolysis in the presence of known characteristic activators and inhibitors of esterases revealed approximately 5% and 20% arylesterase activity in liver and kidney, respectively. The presence of arylesterase activity in these tissues was confirmed by the hydrolysis of paraoxon (E600).

Characterization of rimorphin, a new [Leu] enkephalin-containing peptide from bovine posterior pituitary glands

Life Sciences ◽  
1982 ◽  
Vol 31 (16-17) ◽  
pp. 1849-1852 ◽  
Author(s):  
D.L. Kilpatrick ◽  
A. Wahlström ◽  
H.W. Lahm ◽  
R. Blacher ◽  
E. Ezra ◽  
...  
Keyword(s):  
Posterior Pituitary ◽  
Pituitary Glands

scholarly journals Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1974 ◽  
Vol 143 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Hans Vilhardt ◽  
Derek B. Hope
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Subcellular Fractions ◽  
Neural Lobe ◽  
Bivalent Cations ◽  
Pituitary Glands ◽  
Membrane Bound ◽  
Triton X ◽  
Hydrolysis Of

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg2+-dependent, (b) Ca2+-dependent and (c) Mg2++Na++K+-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg2++Na++K+-dependent enzyme, whereas the Mg2+- and Ca2+-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg2+- and Ca2+-ATPases; however, the activity of the Mg2++Na++K+-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg2+- and Ca2+-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg2+or Ca2+and the other activated only by Ca2+. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.

Partial Purification and Characterization of an ADP Phosphohydrolase from Human Plasma

1971 ◽  
Vol 26 (01) ◽  
pp. 177-191 ◽  
Author(s):  
I Holmsen ◽  
H Holmsen
Keyword(s):  
Substrate Concentration ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Partial Purification ◽  
Low Concentrations ◽  
Isotope Technique ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Plasma Adenosine

SummaryPlasma contains enzymes capable of dephosphorylating ADP, ATP and AMP (adenosine di-, tri- and monophosphate). In platelet-rich plasma these enzymes are important for the regulation of the levels of (platelet-aggregating) ADP and (aggregation-inhibitory) adenosine.Plasma ADPase and ATPase were studied at 1 (µM substrate concentration using an isotope technique. Both enzymes were precipitated from plasma at 45-65% saturation with (NH4)2S04 and emerged together by gel filtration on Sephadex G-200 and from DEAE-Sephadex (0.12-0.20 M Cl-, pH 8.2). In combination these procedures gave 1,500-1,800 times purification of ADPase relative to plasma. The purest fraction contained ATPase, ADPase and AMPase in a 0.17:1.00:2.92 proportion, quite different from their 5.34:1.00:5.34 proportion in plasma. Adenosine deaminase and adenylate kinase were not present in the purest fraction, whereas nucleoside diphosphokinase appeared to be present.The purified ADPase was stimulated by low concentrations of Mg2+ and Mn2+, whereas high concentrations were inhibitory. This inhibition could not be explained by an increase in the ionic strength. Ca2+ and Zn2+ were inhibitory at all concentrations used (0-3 mM). Lineweaver-Burke plots were linear for both ADPase and ATPase in the 0−4 x 10-5 M substrate range, and both enzymes had Km = 1.1 x 10−5 M. Increase of the substrate concentration above 4 x 10−5M gave deviation from MichaelisMenten kinetics, and Eadie-Hofstee plots indicated the presence of “high-Km” ADPase and ATPase. The latter enzymes were not studied.Déphosphorylation of 3H-ADP by purified “low-Km” ADPase was reduced by nonradioactive diphosphates of guanosine, inosine, cytidine and uridine in the same way as when nonradioactive ADP was used. Nonradioactive AMP also reduced dephosphorylation of 3H-ADP, whereas nonradioactive ATP did not.Cyanide, cysteine and tartrate inhibited “low-Km” ADPase whereas p-chloromercuribenzoate, p-chloromercuribenzoesulphonate and N-ethylmaleimide had no effect. EDTA inhibited the enzyme activity in a way that could not be abolished by excess Mg2+. Purified plasma “low-Km” ADPase thus appears to be an unspecific enzyme, as one and the same active site does not seem to distinguish between the base moiety of nucleoside diphosphates, and catalyzes hydrolysis of phosphate esters as well as pyrophosphate bonds. The relation between plasma ADPase and ATPase remains unclear.

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