scholarly journals Properties of phosphatidate phosphohydrolase in rat adipose tissue

1994 ◽  
Vol 301 (3) ◽  
pp. 793-799 ◽  
Author(s):  
S C Jamdar ◽  
W F Cao
Keyword(s):  
Adipose Tissue ◽  
Plasma Membranes ◽  
Lipid A ◽  
Mesangial Cell ◽  
Intraperitoneal Administration ◽  
Subcellular Fractions ◽  
Phosphatidate Phosphohydrolase ◽  
Membrane Bound ◽  
Rat Adipose Tissue ◽  
Triton X

Previously we have identified the presence of two different phosphatidate phosphohydrolase (PPH) activities in rat adipose tissue, based on Mg(2+)-dependency. In the present investigation, we have further characterized these isoenzymes, using both aqueous dispersed and membrane-bound phosphatidate as substrates and differentiated these activities on the basis of both Mg(2+)-dependency and N-ethylmaleimide (NEM)-sensitivity. These two distinguishing criteria gave identical estimates of PPH activities present in the different subcellular fractions. The microsomal and cytosol fractions contained mainly the Mg(2+)-dependent (NEM-sensitive) form, which was inhibited by various thiol reagents, was inactivated by heating at 55 degrees C for 20 min, and was decreased significantly within 2 h after intraperitoneal administration of cystamine (200 mg/kg). Such treatments had no effects on the Mg(2+)-independent (NEM-insensitive) form of PPH, which was mainly located in the plasma membranes, mitochondrial and microsomal fractions. Addition of Lipid A and guanosine 5′-[gamma-thio]triphosphate to the assay mixture had no effect on the PPH activities. The Mg(2+)-independent PPH form, which was thermostable in the intact subcellular fractions, became thermolabile when these fractions were disrupted in the presence of Triton X-100. The present studies demonstrate that: (1) the thermostability is not a satisfactory index to differentiate these isoenzymes; (2) the thiol/disulphide exchange may be involved in the regulation of Mg(2+)-dependent PPH activity; and (3) the PPH isoenzymes do not seem to be under G-protein control in adipose tissue, as reported previously in the mesangial cell line.

BIOCHEMICAL EFFECTS OF INSULIN ON SUBCELLULAR FRACTIONS FROM RAT ADIPOSE TISSUE

1973 ◽  
Vol 71 (4_Suppl) ◽  
pp. S121 ◽  
Author(s):  
E. Pimentel ◽  
C. A. Gonzalez ◽  
F. Gonzalez
Keyword(s):  
Adipose Tissue ◽  
Subcellular Fractions ◽  
Biochemical Effects ◽  
Rat Adipose Tissue

Effects of insulin and glucose on subcellular fractions from rat adipose tissue

1974 ◽  
Vol 11 (3) ◽  
pp. 206-212
Author(s):  
Enrique Pimentel ◽  
Carlos A. González ◽  
Freddy González-Mujica
Keyword(s):  
Adipose Tissue ◽  
Subcellular Fractions ◽  
Rat Adipose Tissue

scholarly journals METABOLIC AND ULTRASTRUCTURAL CHANGES INDUCED IN ADIPOSE TISSUE BY INSULIN

1960 ◽  
Vol 8 (1) ◽  
pp. 83-101 ◽  
Author(s):  
Russell J. Barrnett ◽  
Eric G. Ball
Keyword(s):  
Adipose Tissue ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Morphological Changes ◽  
Ultrastructural Changes ◽  
Cytoplasmic Matrix ◽  
Smooth Membrane ◽  
Metabolic Action ◽  
Rat Adipose Tissue

The addition in vitro of insulin to rat adipose tissue (epididymal) produces marked metabolic changes which may be followed by measurement of the net gas exchange of the tissue. Using this method to monitor the metabolic action of insulin, concomitant observations with the electron microscope on the tissue have been made. These reveal that pronounced morphological changes are induced by insulin. The plasma membranes of the adipose cells become invaginated at many sites to form minute finger-like indentations. Numerous tiny, membrane-bounded vesicles are also present and arranged in relationship to the plasma membrane in such a way as to suggest that their formation occurred when a recessed fold was pinched off. Deeper in the cytoplasm, especially in specimens that had been incubated a longer time, numerous large, smooth, membrane-limited vesicles are seen. Finally, in these incubated specimens the cytoplasmic matrix has lost much of its granular nature, small lipid droplets are frequently found in the cytoplasm and suggestive changes have occurred in mitochondria. In control specimens, incubated without insulin for identical periods of time, indentations and vesicles in the plasma membrane are sparse at best and no vesicles or membrane-bound spaces appear deeper in the cytoplasm. The metabolic and morphologic changes induced by insulin seem to be interdependent events. Both changes appear to be initiated rapidly and concomitantly in the tissue. Both processes are initiated by insulin at concentrations considered to be physiological, 0.004 µg. (100 µunits) per ml. Insulin treated with alkali fails to initiate either process. It is concluded that insulin initiates pinocytosis in rat adipose tissue and the possible significance of this process in the mode of action of insulin is discussed.

scholarly journals Biochemical and functional characterization of the GLUT5 fructose transporter in rat skeletal muscle

1998 ◽  
Vol 336 (2) ◽  
pp. 361-366 ◽  
Author(s):  
Froogh DARAKHSHAN ◽  
Eric HAJDUCH ◽  
Søren KRISTIANSEN ◽  
Erik A. RICHTER ◽  
Harinder S. HUNDAL
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Plasma Membranes ◽  
Functional Characterization ◽  
Protein Band ◽  
Subcellular Fractionation ◽  
Rat Skeletal Muscle ◽  
Reverse Transcription Pcr ◽  
Molecular Approaches ◽  
Rat Adipose Tissue

Previous work has demonstrated that human skeletal muscle and adipose tissue both express the GLUT5 fructose transporter, but to date the issue of whether this protein is also expressed in skeletal muscle and adipose tissue of rodents has remained unresolved. In the present study we have used a combination of biochemical and molecular approaches to ascertain whether rat skeletal muscle expresses GLUT5 protein and, if so, whether it possesses the capacity to transport fructose. An isoform-specific antibody against rat GLUT5 reacted positively with crude membranes prepared from rat skeletal muscle. A single immunoreactive band of approx. 50 kDa was visualized on immunoblots which was lost when using anti-(rat GLUT5) serum that had been pre-adsorbed with the antigenic peptide. Subcellular fractionation of skeletal muscle localized this immunoreactivity to a single membrane fraction that was enriched with sarcolemma. Plasma membranes, but not low-density microsomes, from rat adipose tissue also displayed a single protein band of equivalent molecular mass to that seen in muscle. Reverse transcription–PCR analyses, using rat-specific GLUT5 primers, of muscle and jejunal RNA revealed a single PCR fragment of the expected size in jejunum and in four different skeletal muscle types. Sarcolemmal vesicles from rat muscle displayed fructose and glucose uptake. Vesicular uptake of glucose was inhibited by nearly 90% in the presence of cytochalasin B, whereas that of fructose was unaffected. To determine whether fructose could regulate GLUT5 expression in skeletal muscle, rats were maintained on a fructose-enriched diet for 4 days. This procedure increased jejunal and renal GLUT5 protein expression by approx. 4- and 2-fold respectively, but had no detectable effects on the abundance of GLUT5 protein in skeletal muscle or on fructose uptake in rat adipocytes. The present results show that GLUT5 is expressed in the sarcolemma of rat skeletal muscle and that it is likely to mediate fructose uptake in this tissue. Furthermore, unlike the situation in absorptive and re-absorptive epithelia, GLUT5 expression in insulin-sensitive tissues is not regulated by increased substrate supply.

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.

scholarly journals Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions

1992 ◽  
Vol 285 (1) ◽  
pp. 153-159 ◽  
Author(s):  
I J Cartwright ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Subcellular Fractions ◽  
Apo B ◽  
Sds Page ◽  
Membrane Bound ◽  
Triton X ◽  
Quantitative Immunoblotting

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

scholarly journals A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions

1988 ◽  
Vol 255 (3) ◽  
pp. 999-1005 ◽  
Author(s):  
C Enrich ◽  
O Bachs ◽  
W H Evans
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Specific Protein ◽  
Subcellular Fractions ◽  
Calmodulin Binding ◽  
Overlay Technique ◽  
Protein Partitioning ◽  
Triton X

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol.

scholarly journals Triacylglycerol biosynthesis in the adipose tissue of the obese-hyperglycaemic mouse

1976 ◽  
Vol 158 (2) ◽  
pp. 327-334 ◽  
Author(s):  
S C Jamdar ◽  
D Shapiro ◽  
H J Fallon
Keyword(s):  
Adipose Tissue ◽  
Microsomal Fraction ◽  
Soluble Fraction ◽  
Diacylglycerol Acyltransferase ◽  
Obese Mouse ◽  
Obese Mice ◽  
Triacylglycerol Biosynthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Membrane Bound

Obesity in obese-hyperglycaemic mouse is associated with an increase in number and size of adipocytes. Adipocytes from the obese mouse showed increased incorporation of [14C]acetate and[14C]glucose into triacylglycerol. This increased capacity of triacylglycerol formation was correlated with increased activities of various triacylglycerol-forming enzymes measured in the microsomal fraction of adipose tissue from obese mice. Microsomal fractions from lean and obese mice contained sn-glycerol 3-phosphate acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase. Phosphatidate phosphohydrolase was also detected in the soluble fraction. In the presence of Mg2+, the phosphatidate phsophohydrolase from the soluble and the microsomal fractions was active towards membrane-bound phosphatidate. Among the three enzymes studied here, the increase in Mg2+-dependent phosphatidate phosphohydrolase was most prominent in adipose tissue of obese mice.

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