Phosphorylated and non-phosphorylated forms of catechol O-methyltransferase in rat liver, brain and other tissues

Seven different forms of the enzyme COMT (catechol O-methyltransferase) were found in isolated rat hepatocytes by two-dimensional gel electrophoresis and immunoblotting: five small variants (S-COMT) and two large variants (L-COMT). The identities of these COMT forms were verified by tryptic fingerprinting using MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS, and by amino acid sequencing using ESI–IT–MS/MS (electrospray ionization with ion-trap tandem MS). Analysis of tissue distributions showed that the S-COMT forms were highly expressed in liver and kidney, whereas L-COMT was expressed more strongly in other tissues. Both of the L-COMT forms were found in all of the tissues examined except the heart, which expressed only the most acidic form, and the kidney, which expressed only the most basic form. Subcellular fractionation revealed the presence of both S-COMT and L-COMT in soluble, as well as sedimentable, fractions, suggesting that they should be classified by size rather than (as previously) by localization. Several of the S-COMT forms were N-acetylated, and the two most acidic forms were found to be phosphorylated at Ser260. One of the latter was unique to liver cells; the other was also found in kidney, brain and thymus. Among the non-phosphorylated S-COMT forms, one was ubiquitous, one was found in testis and liver, and a third was found in liver, kidney and thymus. No other phosphorylated sites were found, suggesting that the pI differences distinguishing between the various COMT forms are due to some as yet unidentified structural modification(s).

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The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes

Biochemical Journal ◽

10.1042/bj2370033 ◽

1986 ◽

Vol 237 (1) ◽

pp. 33-39 ◽

Cited By ~ 31

Author(s):

E Fries ◽

I Lindström

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Low Temperatures ◽

Intracellular Transport ◽

Rat Hepatocytes ◽

Subcellular Fractionation ◽

Isolated Rat Hepatocytes ◽

Different Temperatures ◽

Lag Period ◽

Time Courses

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.

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Subcellular fractionation of isolated rat hepatocytes a comparison with liver homogenate

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/0304-4165(80)90338-4 ◽

1980 ◽

Vol 632 (4) ◽

pp. 619-629 ◽

Cited By ~ 39

Author(s):

Geneviève Krack ◽

Odette Gravier ◽

Marcel Roberfroid ◽

Michel Mercier

Keyword(s):

Liver Homogenate ◽

Rat Hepatocytes ◽

Subcellular Fractionation ◽

Isolated Rat Hepatocytes ◽

Isolated Rat

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Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin

Biochemical Journal ◽

10.1042/bj1940155 ◽

1981 ◽

Vol 194 (1) ◽

pp. 155-165 ◽

Cited By ~ 108

Author(s):

C J Kirk ◽

R H Michell ◽

D A Hems

Keyword(s):

Glycogen Phosphorylase ◽

Equilibrium Distribution ◽

Rat Hepatocytes ◽

Subcellular Fractionation ◽

Isolated Rat Hepatocytes ◽

Phosphatidylinositol Metabolism ◽

Maximal Activation ◽

Vasopressin Receptors ◽

Stimulation Of

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.

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Heterogeneity Analysis of the Human Pituitary Proteome

Clinical Chemistry ◽

10.1373/49.10.1740 ◽

2003 ◽

Vol 49 (10) ◽

pp. 1740-1751 ◽

Cited By ~ 28

Author(s):

Xianquan Zhan ◽

Dominic M Desiderio

Keyword(s):

Mass Spectrometry ◽

Ion Trap ◽

Thyroid Hormone Receptor ◽

Quadrupole Ion Trap ◽

Hormone Regulation ◽

Human Pituitary ◽

Two Dimensional Gel Electrophoresis ◽

Ionization Time ◽

Flight Mass Spectrometry ◽

Thyroid Hormone Receptor Β

Abstract Background: A human proteome is relatively dynamic compared with its corresponding genome. Our aim was to study the heterogeneity of a human pituitary proteome as a function of gender, age, and race. Methods: Pituitary control tissues (n = 8) were used to extract proteins; each control tissue was analyzed (n = 3–5) with two-dimensional gel electrophoresis (2DGE) and PDQuest software. We obtained 30 high-resolution 2DGE gels and conducted a comparative analysis as a function of gender, age, and race. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry were used to characterize the protein in each differential spot. Results: We detected ∼1000 protein spots in each 2DGE map, and 51 differential spots (7 differing with gender, 17 with age, 15 with race, and 12 with the coeffect of age and race). Among those 51, we characterized 28 proteins [5 differing with gender, 8 with age, 6 with race, 8 with the coeffect of age and race, and 1 (somatotropin chain 1) with all of these]. Somatotropin was related to gender, age, and race, and prolactin was higher in females than males. The differentially expressed proteins that were related to age were mainly those proteins associated with cell growth, proliferation, differentiation, apoptosis, and death; those proteins showed no difference with gender and race. Age and race affected some proteins associated with hormone regulation (e.g., follistatin, thyroid hormone receptor β-2, adenylate cyclase-inhibiting Gα protein). Conclusions: A heterogeneity exists in the human pituitary proteome as a function of gender, age, and race. These findings will serve as a basis for our comparative proteomics studies of human pituitary adenomas.

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Binding of concanavalin A to isolated hepatocytes and its effect on uptake and degradation of asialo-fetuin by the cells

Biochemical Journal ◽

10.1042/bj1900697 ◽

1980 ◽

Vol 190 (3) ◽

pp. 697-703 ◽

Cited By ~ 14

Author(s):

H Tolleshaug ◽

M Abdelnour ◽

T Berg

Keyword(s):

Cell Density ◽

Concanavalin A ◽

Binding Capacity ◽

Rat Hepatocytes ◽

Subcellular Fractionation ◽

Isolated Hepatocytes ◽

Isolated Rat Hepatocytes ◽

Isolated Rat

1. The binding of 125I-labelled concanavalin A to isolated rat hepatocytes was studied at temperatures between 4 degrees C and 37 degrees C. At the latter temperature, concentrations of concanavalin A from 0.01 to 0.4 mg/ml were used. In all of these experiments, binding reached a plateau after 40—60 min, when 28—35% of the concanavalin A added was bound to the cells (cell density 8 × 10(6) cells/ml). 2. The rate of uptake of 125I-labelled asialo-fetuin by the hepatocytes was lowered to 30% of control values when the cells were preincubated with 0.1 mg of concanavalin A/ml. This decrease could be accounted for by a decrease in the rate of binding of asialo-fetuin to the beta-galactoside receptor of the cells. The binding capacity of the cells was not influenced by preincubation with concanavalin A. 3. Degradation of asialo-fetuin was decreased only if concanavalin A was present during the uptake of asialo-fetuin by the cells. Subcellular fractionation revealed that concanavalin A lowered the rate of entry of endocytosed asialo-fetuin into the lysosomes. The effect of concanavalin A on degradation is distinct from its effect on the rate of uptake of asialo-fetuin by hepatocytes.

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