Paul A. Weinhold

Activity and properties of CTP: cholinephosphate cytidylyltransferase in adult and fetal rat lung.

Cholinephosphate cytidylyltransferase (CTP : cholinephosphate cytidylyltransferase, EC 2.7.7.15) is located in both the microsomal and supernatant fractions of adult lung when the tissue is homogenized in 0.145 M NaCl. The activity is located predominantly in the supernatant fraction in fetal lung. Cholinephosphate cytidylyltransferase in the supernatant from fetal lung is stimulated 4- to 6-fold by the additions of total lung lipid. Serine phosphoglycerides and inositol phosphoglycerides specifically caused stimulation whereas choline phosphoglycerides and ethanolamine phosphoglycerides produced no stimulation. Lysophosphatidylcholine cause some stimulation, but only at high concentrations. A number of detergents were investigated. All produced inhibition except for the ampholytic detergent, miranol H2M which was not inhibitory. None of the detergents produced any stimulation of activity. Cytidylyltransferase activity in fetal lung when assayed in the absence of lipid is about 25% of the adult. The activity when assayed in the presence of lipid is equal or slightly higher than adult levels. The activity, measured without added phospholipid, increases 5- to 6-fold within 12 h after birth, to values higher than in the adult. The activity, measured in the presence of phospholipid, increased almost linearly from -2 day until +1 day. There is an inverse relationship between the concentration of phospholipid in the fetal lung supernatant and the degree of lipid stimulation. Chromatographic experiments with Biogel A 1.5 columns have shown that cytidylyltransferase can exist in two molecular sizes, a small molecular size that requires phospholipid for activity, and a larger molecular weight species which does not require the addition of phospholipid for activity. Fetal lung has a higher proportion of the low molecular weight form than adult lung. The small molecular weight species can be converted to the larger molecular weight form by the addition of phospholipids.

Synthesis and prostaglandin E2-induced secretion of surfactant phospholipid by isolated gastric mucous cells.

Lipids, particularly surface-active phospholipids, have been proposed to provide an important protective barrier in the gastric mucosa. The predominant surface-active phospholipid in the pulmonary surfactant complex is dipalmitoylphosphatidylcholine. To determine whether the gastric epithelium synthesizes and secretes this phospholipid, primary cultures of canine gastric mucous cells isolated by counterflow elutriation were studied. During the 24-hour period of culture, the gastric mucous cells incorporated 3H-choline into phosphatidylcholine, with dipalmitoylphosphatidylcholine representing 13.8% +/- 0.6% of the phosphatidylcholine synthesized. When mucous cell preparations with greater chief cell contamination were studied, they incorporated significantly less precursor into dipalmitoylphosphatidylcholine. Administration of prostaglandin E2, a cytoprotective agent, to the cultured mucous cells for 1 hour led to a significant increase in phosphatidylcholine release, reaching a maximum of 120.4% +/- 4.2% (P less than 0.001) at 10(-6) mol/L. No significant stimulation of phospholipid release by prostaglandin E2 was seen in the fractions containing a greater proportion of chief cells. To further establish the relationship between mucin and phospholipid secretion, two gastric cancer cell lines, Hs746T and KATO III, were studied. Using immunocytochemical and biochemical techniques, mucin synthesis and secretion were confirmed by these cell lines. The Hs746T cells were significantly more active in the secretion of both mucin and phospholipid than the KATO III cells. The Hs746T line secreted 5.7-fold more mucin and 7.3-fold more phospholipid than KATO III cells during a 24-hour period of culture. The association between mucin and phospholipids in an aqueous solution was also studied. Purified mucin in the concentration of 0.5-2 mg/mL of glycoprotein led to a significant dose-dependent increase in phospholipid solubility, suggesting the formation of a glycoprotein-phospholipid complex. The current studies indicate that the gastric mucous cell is the source of surfactant phospholipids as well as mucin. The synthesis and release of mucin and phospholipid are functions of the mucous cell that play a critical role in the primary defense of gastric epithelium.

EVIDENCE FOR A REGULATORY ROLE OF CTP : CHOLINE PHOSPHATE CYTIDYLYLTRANSFERASE IN THE SYNTHESIS OF PHOSPHATIDYLCHOLINE IN FETAL LUNG FOLLOWING PREMATURE BIRTH

The sequence of reactions which function to incorporate choline into phosphatidylcholine was investigated in lung from fetuses following premature delivery. The rate of [methyl-14C]choline incorporation by rat lung slices into phosphatidylcholine increases following premature delivery at both 20 and 21 days gestation. The increase in choline incorporation is primarily due to an increased specific activity of phosphorylcholine resulting from a decreased pool size of phosphorylcholine. The decrease in the concentration of phosphorylcholine following premature delivery is apparently caused by an increased activity of cytidylyltransferase which leads to an increase in the conversion of phosphorylcholine to phosphatidylcholine. The total activity of choline kinase, cytidylyltransferase, cholinephosphotransferase and phosphatidate phosphohydrolase did not change significantly. However, the cytidylyltransferase activity in the microsome fraction increased following premature delivery at 20 and 21 days gestation. The amount of cytidylyltransferase in the H form in the cytosol fraction increased following premature delivery at 21 days gestation but not at 20 days gestation. The results are interpreted to indicate that the active form of cytidylyltransferase in lung cells is the membrane-bound enzyme and this form increases following birth resulting in an increased synthesis of phosphatidylcholine.

Tumor localizing agents. IX. Radioiodinated cholesterol

19-Iodocholesterol- 125 I was synthesized for study as a possible agent for photoscanning the adrenal gland and associated tumors. In contrast to previous radioiodinated steroids, it was found to be much less prone to rapid in vivo deiodination. Preliminary tissue distribution studies and biochemical analyses have revealed a marked similarity in the behavior of the radioiodinated derivative with the natural steroid. The concentration of radioactivity in the adrenal cortex of dogs at 48 hours was found to greatly exceed that found in other organs.

Characterization of cytosolic forms of CTP: choline-phosphate cytidylyltransferase in lung, isolated alveolar type II cells, A549 cell and Hep G2 cells

The subcellular forms of cytidylyltransferase (EC 2.7.7.15) in rat lung, rat liver, Hep G2 cells, A549 cells and alveolar Type II cells from adult rats were separated by glycerol density centrifugation. Cytosol prepared from lung, Hep G2 cells, A549 cells and alveolar Type II cells contained two forms of the enzyme. These species were identical to the L-Form and H-Form isolated previously from lung cytosol by gel filtration. Liver cytosol contained only the L-Form. Rapid treatment of Hep G2 cells with digitonin released all of the cytoplasmic cytidylyltransferase activity. The released activity was present in both H-Form and L-Form. The molecular weight of L-Form was determined from sedimentation coefficients and Stokes radius values to be 97,690 +/- 10,175. Thus, the L-Form appears to be a dimer of the Mr 45,000 catalytic subunit. The f/f degrees value of 1.5 indicated that the protein molecule has an axial ratio of 10, assuming a prolate ellipsoid shape. The estimated molecular weight of the H-Form was 284,000 +/- 25,000. The H-Form was dissociated into L-Form by incubation of cytosol at 37 degrees C. Triton X-100 (0.1%) and chlorpromazine (1.0 mM) also dissociated the H-Form into L-Form. Western blot analysis indicated that both forms contained the catalytic subunit. An increase in Mr 45,000 subunit coincided with the increase in cytidylyltransferase activity in L-Form, which resulted from the dissociated of H-Form. The L-Form was dependent on phospholipid for activity. The H-Form was active without lipid. Phosphatidylinositol was present in the H-Form isolated from Hep G2 cells. The phosphatidylinositol dispersed when the H-Form was dissociated into L-Form. Phosphatidylinositol and phosphatidylglycerol cause L-Form to aggregate into a form similar to H-Form. Phosphatidylcholine/oleic acid (1:1 molar ratio) and oleic acid also aggregated the L-Form. Phosphatidylcholine did not produce aggregation. We conclude that the H-Form is the active form of cytidylyltransferase in cytoplasm. The H-Form appears to be a lipoprotein consisting of an apoprotein (L-Form dimer of the Mr 45,000 subunit) complexed with lipids. A change in the relative distribution of H-Form and L-Form in cytosol would alter the cellular activity and thus may be important in the regulation of phosphatidylcholine synthesis.

Activation of CTP : Phosphocholine cytidylyltransferase in rat lung by fatty acids

CTP : phosphocholine cytidylyltransferase activity exists in both the microsome and cytosol fractions of adult lung, 36 and 59%, respectively. Although these enzyme activities are stimulated in vitro by added lipid activators (i.e. phosphatidylglycerol), there are significant levels of activity in the absence of added lipid. We have removed endogenous lipid material from microsome and cytosol preparations of rat lung by rapid extraction with isopropyl ether. The extraction procedure did not cause any loss of cytidylyltransferase activity in the cytosol. After the extraction the enzyme was almost completely dependent upon added lipid activator. Isopropyl ether extraction of microsome preparations produced a loss of 40% of the cytidylyltransferase activity, when measured in the presence of added phosphatidylglycerol. Lipid material extracted into isopropyl ether restored the cytidylyltransferase activity in cytosol. The predominant species of enzyme activator in the isopropyl ether extracts was fatty acid. A variety of naturally occurring unsaturated fatty acids stimulated the cytidylyltransferase to the same extent as phosphatidylglycerol. Saturated fatty acids were inactive.

Activity and some properties of choline kinase, cholinephosphate cytidyltransferase and choline phosphotransferase during liver development in the rat

Abstract The activity of choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) cholinephosphate cytidyltransferase (CTP:cholinephosphate cytidyltransferase, EC 2.7.7.15) and cholinephosphotransferase (CDP-choline:1,2-diacylglycerolcholine-phosphotransferase, EC 2.7.8.2) was determined during the development of the liver in the rat. Choline kinase activity was located in the 100000 × g supernatant and is slightly lower than adult in — 5 day fetal liver. The activity increases to almost 2 times the adult prior to birth and declines to adult levels shortly after birth. The cholinephosphate cytidyltransferase is found at all ages in both the 100000 × g supernatant and in microsomes. The distribution of activity between these two fractions varies with the age of the animal. The activity in the supernatant fraction from — 5 day fetal is almost twice the adult values, whereas the activity in the microsome fraction from — 5 day fetal does not significantly differ from the adult. The activity in both fractions increases following birth. The activity of cholinephosphotransferase in the presence of exogenous diglyceride was about 10% of the adult values in — 5 day fetal liver. The activity increased slightly prior to birth but did not approach the adult levels until 8 days after birth. The addition of diglyceride to the assays did not increase the enzyme activity from fetal liver but caused a 2–3-fold increase in enzyme activity from 5 day and older animals. The developmental activities of the three enzymes are compared to the previously obtained developmental pattern for the incorporation of [Me- 14 C]-choline into choline phosphoglycerides by liver slices.

Age-related decline in protein synthesis in the rat parotid gland

Abstract This study was undertaken to determine whether protein synthesis declines with age in parorid salivary glands. We have examined the level of incorporation of [3H]-leucine into protein and the metabolism of [U-14C]-D-glucose in 2-, 12-, 18-, 24- and 30-month-old rats. The rate of incorporation of [3H]-leucine into perchloric acid insoluble protein by gland slices decreases progressively with age from 138.43±5.43 nmole/mg DNA/h at 2 months to 109.77±6.88, 97±4.68, 71.62±4.13 and 58.08±5.61 nmole/mg DNA/h at 12, 18, 24 and 30 months, respectively. The amount of radioactivity in the perchloric acid soluble fraction remains relatively stable at all ages. The ability of gland slices to oxidize [U-14C]-D-glucose to 14CO2 and to incorporate radioactivity into the total lipid does not change with age, whereas the incorporation of radioactivity into protein decreases significantly from 45.06±3.55 at 2 months to 21.75±1.30 nmoles glucuse/mg DNA/h at 24 months. Histologically, no significant change is detected in the proportion of secretory cells to ductal cells in the different age groups. In radioautographs of the glands incubated with [3H]-leucine, the distribution of cells with grains is similar in all age groups. Lipid droplets occur at the periphery of the lobules and a large number of lipofuscin granules are present in secretory acinar cells of 12-month-old and older rats when viewed in the electron microscope. However, the structural integrity in the cellular organelles is maintained in the acinar cells of all age groups. These results indicate that the ability of parotid gland cells to synthesize protein decreases with age. The decrease reflects specific changes in protein synthesis and not an overall decline in cellular metabolism or a decrease in the number of cells which incorporate leucine.

COMPARISON OF THE PHOSPHOLIPID REQUIREMENTS AND MOLECULAR FORM OF CTP : PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE FROM RAT LUNG, KIDNEY, BRAIN AND LIVER

The cytidylyltransferase activity in fresh cytosol from different tissues of the rat was measured in the absence and presence of phosphatidylglycerol. In all cases addition of this lipid produced large increases in enzyme activity. Agarose gel (A-5.0) filtration profiles of the enzyme activities indicated that the L-form of the enzyme (190 000 molecular weight) predominated in liver, brain, kidney, and fetal lung. However, adult lung cytosol contained 70--80% of the activity in the H-form (molecular weight greater than or equal to 5 x 10(6)). Removal of phospholipid material from the alveolar spaces by lavage produced a significant reduction of the H-form of the enzyme in the cytosol fraction. The L-form of the cytidylyltransferases from fetal lung and adult liver, kidney, and brain all possess the same specificities for activation by phospholipids in vitro. In all cases, phosphatidylglycerol was the most potent activator at 0.2 mM. Lysophosphatidylethanolamine stimulated enzyme activity, whereas lysophosphatidylglycerol was a potent inhibitor. These studies implicate the role of acidic phospholipids in the regulation of cytidylyltransferase activity in vivo and the existence of a common L-form of the enzyme in serveral tissues of the rat.

[29] Choline-phosphate cytidylyltransferase

Publisher Summary Choline-phosphate cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase) catalyzes a major rate-determining step in the biosynthesis of phosphatidylcholine in mammalian cells. Both cytosolic and membrane fractions contain choline-phosphate cytidylyltransferase activity. Enzyme activity is determined by measuring the formation of radioactive CDPcholine from phospho[methyl- 14 C]choline. Two methods have been used to separate CDPcholine from phosphocholine: adsorption of CDPcholine by charcoal or separation of CDPcholine from phosphocholine by thin-layer chromatography. The charcoal adsorption method is rapid, sensitive, and reproducible. The ability to perform many assays (50–100) per day is an additional advantage. A variety of lipids have been used by investigators for the assay of cytidylyltransferase. It has been a common practice to include lipid in assays involving soluble forms of cytidylyltransferase. The molecular and kinetic properties of cytidylyltransferase are discussed this chapter.