Search PubMed⌕ Search

Biomedical subjects

W W Wells

Publications and source records attributed to W W Wells.

At least 55 records · Page 3Linked to original sources
PubMed1984

Solubilization and reconstitution of a nuclear envelope-associated ATPase. Synergistic activation by RNA and polyphosphoinositides.

Treatment of isolated rat liver nuclear envelopes with 1% Triton X-100 solubilized 20-30% of the nuclear envelope protein and 85% of the ATPase activity. Chromatography on DEAE-Sepharose in 1% Triton X-100 at pH 7.5 removed all of the chemically measurable phospholipid from the bound ATPase activity; however, endogenously synthesized phosphatidylinositol [4-32P]phosphate (PIP) co-chromatographed with the ATPase activity on this column. Further purification by chromatography on heparin-agarose removed all of the [32P]PIP and RNA from the ATPase; however, the recovery of ATPase activity from this column was very low. RNA, polyadenylic acid, and polyguanylic acid stimulated the delipidated ATPase activity 4-6-fold. This activity was not further stimulated by the addition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid, but was stimulated 2-fold by the addition of phosphatidylinositol. Addition of 40 microM PIP and 50 micrograms of RNA/ml resulted in a 25-fold stimulation of basal ATPase activity. Phosphatidylinositol 4,5-bisphosphate, in the presence of RNA, was also able to stimulate ATPase activity but to a lower extent than that of PIP. Therefore, the nuclear envelope-associated ATPase appears to require interaction with a polynucleotide and PIP to express full activity. PIP, which is actively metabolized in the nuclear envelope, may therefore be involved in the regulation of the activity of this enzyme.

Adenosine Triphosphatases↗
PubMed1984

The microtubule-associated nucleoside diphosphate kinase.

Microtubule protein prepared by cycles of assembly-disassembly contains a nucleoside diphosphate kinase (NDP kinase) activity. We have isolated the NDP kinase responsible for this activity from twice-polymerized bovine brain microtubule protein by a five-step chromatographic procedure. The molecular weight of this enzyme was 103,000 +/- 7,000 daltons as determined by sedimentation equilibrium experiments performed with a Beckman Airfuge. A doublet of subunit bands with molecular masses of about 18,000 daltons was detected by silver staining after gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this preparation. We conclude that the enzyme is a hexamer, although we cannot identify the mix of subunits. We were able to isolate only nanogram quantities of this enzyme, too little for extensive studies, so we isolated the enzyme directly from bovine brain without a preliminary microtubule protein isolation. The whole-brain NDP kinase was isolated by the same chromatographic steps as the enzyme from microtubule protein preparations. Both enzymes had a doublet of subunits at the same molecular weights and both were the same isozyme, chromatofocusing at a pH of 8.0. Both enzymes had similar kinetic properties and similar thermal inactivation profiles. These similar properties of the two enzymes suggest that they are identical. Both subunits of NDP kinase could be reversibly phosphorylated by ATP. Phosphorylation of the native enzyme created multiple, more acidic forms that retained activity. The isolation of this NDP kinase, which can copurify with microtubule protein through cycles of assembly-disassembly, will facilitate future studies on the role of this enzyme in the mechanism and regulation of microtubule assembly.

Animals↗
PubMed1984

Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes.

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.

Animals↗
PubMed1984

Subcellular site and mechanism of vasopressin-stimulated hydrolysis of phosphoinositides in rat hepatocytes.

The intracellular site of vasopressin-induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 s of vasopressin treatment of hepatocytes prelabeled with 32Pi, the levels of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4,5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%, then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P] PI-P and [32P]PI-P2 were observed in the other subcellular fractions, and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol in the plasma membrane decreased by 15% after 45 s of vasopressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes, it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphosphoinositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (200 nM) of free calcium when assayed at low ionic strength, but the calcium requirement shifted to micromolar concentrations under isosmotic, intracellular-like, ionic conditions. Addition of vasopressin (200 nM) to the 32P-labeled plasma membranes stimulated the breakdown of 20% of the [32P]PI-P2 present in the plasma membranes in 1 min when assayed under isosmotic conditions in the presence of 2 nM MgCl2 and approximately 200 nM free calcium. This suggests that the phosphoinositide-specific phosphodiesterase is not active under normal cellular conditions, but is activated upon the addition of vasopressin to the intact cell.

Animals↗
PubMed1984

Characterization of a phosphatidylinositol 4-phosphate-specific phosphomonoesterase in rat liver nuclear envelopes.

Incubation of rat liver nuclear envelopes with [gamma-32P]ATP resulted in the synthesis of phosphatidylinositol-[4-32P]phosphate (PIP). Degradation of endogenously labeled PIP was observed upon the dilution of the labeled ATP with an excess of unlabeled ATP. This degradation was most rapid in the presence of EDTA, and was inhibited by MgCl2 and CaCl2. To further characterize the degradative activity, phosphatidylinositol[4-32P]phosphate and phosphatidylinositol [4,5-32P]bisphosphate (PIP2) were synthesized and isolated from erythrocyte plasma membranes. The 32P-labeled phospholipids were then resuspended in 0.4% Tween 80, a detergent that did not inhibit degradation of endogenously labeled PIP, and mixed with nuclear envelopes. [32P]PIP and [32P]PIP2 were degraded at rates of 2.25 and 0.04 nmol min-1 mg nuclear envelope protein-1, respectively. Only 32P was released from phosphatidyl[2-3H]inositol-[4-32P]phosphate, indicating that hydrolysis of PIP was due to a phosphomonoesterase activity (EC 3.1.3.36) in nuclear envelopes. Similarly, anion-exchange chromatographic analysis of the water-soluble products released from [32P]PIP indicated that inorganic phosphate was the sole 32P-labeled product. Hydrolysis of PIP was most rapid at neutral pH, and was not affected by inhibitors of acid phosphatase or alkaline phosphatase. Hydrolysis of PIP was also not inhibited by nonspecific phosphatase substrates, such as glycerophosphate, p-nitrophenylphosphate, AMP, or glucose 6-phosphate. Hydrolysis was stimulated by putrescine, and was inhibited by inositol 2-phosphate, spermidine, spermine, and neomycin.

Animals↗
PubMed1984

Inositol as a lipotropic agent in dairy cattle diets.

Fatty liver syndrome or hepatic lipidosis (HL) is a condition thought to contribute to an increased incidence of peripartum disease, reduced response to therapy and decreased fertility in dairy cows. This syndrome is characterized by excess triglyceride (TG) accumulation in the liver and apparent decreased hepatic lipoprotein output. In lactating rats, a similar condition results from feeding an inositol-deficient diet. It is also characterized by excess hepatic TG accumulation and decreased hepatic lipoprotein output. Myo-inositol is a necessary component of the phospholipid phosphatidyl-inositol, which is an important membrane constituent. Myo-inositol occurs in feed mainly as the inositol hexaphosphate phytic acid. Phytic acid is undigestible by the monogastric but rumen phytases are assumed to adequately hydrolyze it. In early lactation dairy cows, lipid mobilization is intense, and the myo-inositol requirement may exceed the dietary supply or availability. Myo-inositol is being tested in a field trial as a potential lipotropic agent for dairy cows. Preliminary results suggest no lipotropic benefit from added myo-inositol.

Animals↗
PubMed1983

Phosphorylation of rat liver nuclear envelopes. I. Characterization of in vitro protein phosphorylation.

Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.

Adenosine Triphosphatases↗
PubMed1983

Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation.

Incubation of nuclear envelopes isolated from normal rat liver with [gamma-32P]ATP resulted in the rapid labeling of chloroform-soluble products. These products were identified as phosphatidic acid (PA), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-bisphosphate (TPI) based on their chromatographic mobilities on Silica Gel H and cellulose thin layer plates, and by analysis of their deacylation products by high pressure liquid chromatography. The extent of phosphorylation of these products was dependent on the method used to isolate the nuclear envelopes. Relatively gentle isolation methods, such as lysis of purified nuclei with heparin or digestion with deoxyribonuclease I, produce nuclear envelopes which possess significantly higher lipid kinase activity than do envelopes isolated by sonication. Incorporation of 32P into PA, DPI, and TPI in 2 min under standard assay conditions was 13, 152, and 22 pmol/mg of membrane protein, respectively. Degradation of labeled DPI and TPI was evident after 2-5 min of incubation. Nuclear envelope-associated diacylglycerol kinase, phosphatidylinositol kinase, and DPI kinase are characterized with regard to their pH and Mg2+ requirements. The effects of metals, phospholipids, and sulfhydryl reagents on these kinase activities are also described.

1-Phosphatidylinositol 4-Kinase↗
PubMed1983

Identification of phosphatidylinositol kinase in rat liver lysosomal membranes.

Liver lysosomes from Triton-injected or normal rats were found to rapidly incorporate 32P from [gamma-32P]ATP into a lipid component of the membrane, in vitro. The lipid was identified as phosphatidylinositol 4-phosphate based on its chromatographic behavior on Silica Gel H thin layer plates as compared with standard phosphoinositides. The deacylation product, glyceryl-phosphorylinositol phosphate, was compared with standards in chromatographic and electrophoretic systems to further substantiate the identification of the radioactive material. A trace of phosphatidylinositol 4,5-bisphosphate was also found. The properties of the lysosomal membrane phosphatidylinositol kinase were examined using both endogenous lipid and exogenous phosphatidylinositol as substrate. The enzyme was active at neutral pH in the presence of 20 mM MgCl2. The addition of 0.4% Triton X-100 stimulated the enzyme activity toward endogenous substrate, and the highest activity was observed in the presence of detergent and 1 mM phosphatidylinositol. Degradation of the product was seen only in the presence of Triton X-100. The specific activity of the lysosomal phosphatidylinositol kinase is comparable to the detergent-stimulated activity of liver microsomes and plasma membrane, the previously recognized sources of this enzyme in the liver cell.

1-Phosphatidylinositol 4-Kinase↗
PubMed1982

Characterization of endogenous protein phosphorylation in isolated rat liver lysosomes.

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.

Animals↗
PubMed1981

Induction of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes. Requirement for insulin and dexamethasone.

To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.

Animals↗
PubMed1979

myo-Inositol deficiency: studies on the mechanism of lactation-dependent fatty liver formation in the rat.

Intravenous administration of [1-14C]palmitate to 8-day lactating rats fed a myo-inositol supplemented or deficient diet resulted in rapid labeling of liver triglycerides and phospholipids. Compared with myo-inositol deficient rats, those supplemented with myo-inositol showed a greater loss of isotope from liver triglycerides with a more rapid appearance of isotope in serum triglyceride. Loss of 14C from liver phospholipids was similar for both groups, whereas the appearance of labeled phospholipids in serum was slightly greater for myo-inositol supplemented controls compared with myo-inositol deficient rats. The labeling pattern of liver microsomal triglycerides and phospholipids of the two dietary groups was similar; however, liver microsomal protein was significantly reduced in myo-inositol deficient rats relative to the control group. Concurrent administration of Triton WR-1339 with [1-14C]palmitate resulted in significantly less label accumulation in serum triglycerides of myo-inositol deficient rats compared with myo-inositol supplemented rats. Labeled triglycerides in whole liver and in liver microsomes of myo-inositol supplemented rats turned over more rapidly than those of myo-inositol deficient rats while no significant difference was noted for the [14C]palmitate labeled phospholipid of either source. The incorporation of [guanido-14C]arginine into total liver and serum protein 1 hour after injection of the precursor was similar whether the 14-day lactating dams were myo-inositol supplemented or deficient, but total serum protein specific radioactivity of myo-inositol deficient rats was 66% that of myo-inositol supplemented rats. Thus, the significantly reduced release of hepatic triglycerides appears to be the cause of the fatty liver observed during lactation-dependent myo-inositol deficiency.

Animals↗