Phosphatidate phosphatase: activity and properties in fetal and adult rat lung.

The purpose of this work is to compare the properties of phosphatidate phosphatase (L-alpha-phosphatidate phosphohydrolase, EC 3.1.3.4) in fetal and adult rat lung and to establish the developmental profile of activity measured under optimal conditions. The maximal pH of 6.0--7.0 and the inhibition by fluoride, Ca2+ and detergents were simialr for both adult and fetal. Phosphatidate phosphohydrolase activity was located in both mitochondria and microsomes. The localizations of marker enzymes indicated that the activity in these subfractions was not a result of cross contaminations. Very low activity was detected in the supernatant fraction and no Mg2+ requirement was demonstrable. The activity in the particulate fraction was about 50% of the adult from 18 day gestation until birth. Following birth, the activity rapidly increased to adult levels. Dipalmitoyl, dioleoyl and diacyl glycerol 3-phosphates are all utilized well as substrates. 1,2-dipalmitoyl-sn-glycerol 3-phosphate was hydrolyzed faster under maximal conditions. The velocity-substrate curves tended to be sigmoidal, particularly when 1,2-dipalmitoyl-sn-glycerol 3-phosphate was the substrate. Estimated apparent Km values of 0.02--0.03 mM were obtained for fetal and adult preparations.     

Ethanolaminephosphotransferase in rat lung: selectivity for endogenous and exogenous diacylglycerol

Ethanolaminephosphotransferase (EC 2.7.8.1) activity was determined in lung microsomes using diacylglycerols generated endogenously from [14C]glycerol 3-phosphate and different mixtures of fatty acids. Ethanolaminephosphotransferase used endogenously generated dipalmitoylglycerol better than dioleoylglycerol. The apparent Km and the reaction rates for four different endogenously generated mixtures were the same (16 nmol/mg microsomal proteins). The apparent Km values for CDP-ethanolamine were the same (0.26 mm) for endogenously generated dipalmitoylglycerol and dioleoylglycerol. The amount of diacylglycerol generated in microsomes was 2-3-times the apparent Km for diacylglycerol. Dipalmitoylglycerol, supplied exogenously as a Tween 20/phosphatidylglycerol emulsion, was nearly twice as active as dioleoylglycerol. Both dipalmitoylglycerol and dioleoylglycerol were more active as substrates when emulsions were made with phosphatidylglycerol/Tween 20 than with Tween 20 alone. The results suggest that ethanolaminephosphotransferase in lung is relatively nonselective for molecular species of diacylglycerol. In addition, the results suggest that the concentration of diacylglycerol and the physical state in which it is presented to the enzyme can affect the apparent selectivity of ethanolaminephosphotransferase for diacylglycerols.     

Developmental differences in activation of cholinephosphate cytidylyltransferase by lipids in rabbit lung cytosol

Lung cytosolic cholinephosphate cytidylyltransferase is activated by lipids. We examined the lipid activation pattern as a function of development in rabbit lung from 27 days gestation through term (31 days) and in the adult. The enzyme in both the fetal and adult cytosol was dependent on lipids for activity. Extraction of the cytosol with acetone/butanol virtually abolished cytidylyltransferase activity, but the activity could be restored on addition of lipids extracted with chloroform/methanol from additional cytosol. Cytosolic phospholipids from the fetal lung reactivated cytidylyltransferase but both neutral lipids and phospholipids from the adult were required. The lipids had the same effect on cytidylyltransferase activity in delipidated cytosol from either the fetus or adult so the difference in activation pattern was attributable to the lipids rather than the protein. There was a shift from the fetal to the adult lipid activation pattern as development progressed. Further, there was a significant correlation between cytidylyltransferase activities in intact cytosols from developing lung and activities in delipidated cytosol in the presence of lipids from the same animals. Although these data suggest that lipids regulate cytosolic cytidylyltransferase activity in developing lung their physiological significance remains to be established.     

The supply of both CDP-choline and diacylglycerol can regulate the rate of phosphatidylcholine synthesis in HeLa cells

The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was measured in HeLa cells permeabilized with 0.125 mg digitonin/mL. The rate of phosphatidylcholine formation was influenced by the concentration of CDP-choline in the medium. The CDP-choline:1,2-diacylglycerol cholinephosphotransferase in permeabilized cells showed a Km of 88 microM for CDP-choline. A similar Km value of 104 microM was found for cholinephosphotransferase in microsomes isolated from HeLa cells when assayed in the presence of 2.4 mM dioleoylglycerol. In the absence of added diacylglycerol, the Km for CDP-choline for the microsomal cholinephosphotransferase was only 38 microM. The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was stimulated by the supply of diacylglycerol in both HeLa cells and isolated microsomes. A 2.4 mM dioleoylglycerol suspension increased cholinephosphotransferase activity fourfold in microsomes. The digitonin-treated cells were impermeable to the dioleoylglycerol suspension. Incubation of permeabilized cells with 150 microM acyl-CoA and 0.8 mM glycero-3-phosphate tripled cellular diacylglycerol levels, causing a doubling in the rate of phosphatidylcholine synthesis. A similar incubation of microsomes with acyl-CoA stimulated phosphatidylcholine synthesis twofold. Furthermore, incubation of microsomes with [3H]diacylglycerol and [14C]CDP-choline showed that both of the substrates were incorporated into phosphatidylcholine at the same rate. This result suggests that the stimulatory effects on cholinephosphotransferase arise from increases in the availability of substrates rather than activation of the enzyme. These results suggest that both in the permeabilized cells and in isolated membranes, the biosynthesis of phosphatidylcholine can be limited by both CDP-choline and diacylglycerol.     

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5-10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10-20 min), the rates were comparable.     

Characterization of sheep liver and lung microsomal ethylmorphine N-demethylase

Ethylmorphine N-demethylase activity of the sheep liver and lung microsomes was reconstituted in the presence of solubilized microsomal cytochrome P-450, NADPH-cytochrome c reductase and synthetic lipid, phosphatidylcholine dilauroyl. The Km of the lung microsomal ethylmorphine N-demethylase was calculated to be 4.84 mM ethylmorphine from its Lineweaver-Burk graph and lung enzyme was inhibited by its substrate, ethylmorphine, when its concn was 25 mM and above, reaching to 67% inhibition at 50 mM concn. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear. From these graphs, two different Km values were calculated for the liver enzyme as 4.17 mM and 0.40 mM ethylmorphine. Ethylmorphine N-demethylase activities of both liver and lung microsomes were inhibited by NiCl2, CdCl2 and ZnSO4. Ethylalcohol inhibited N-demethylation of ethylmorphine in lung and liver microsomes. Acetone (5%) slightly enhanced the N-demethylase activity of the liver enzyme, whereas 5% acetone completely inhibited the lung enzyme. Phenylmethylsulfonyl fluoride at 0.10 mM and 0.25 mM concn had no effect on liver enzyme activity, while at these concns, it inhibited the activity of the lung enzyme by about 35%.     

The role of phosphatidylglycerol in the activation of CTP:phosphocholine cytidylyltransferase from rat lung.

The reaction catalyzed by CTP:phosphocholine cytidylyltransferase in the reverse direction, i.e. the formation of CTP and phosphocholine from CDP-choline and pyrophosphate, is slightly faster than the reaction in the forward direction. The reverse reaction is optimal at 2 mM pyrophosphate and 6 mM Mg2+, in both fetal and adult preparations. The apparent substrate Km values for phosphocholine, CDP-choline, and pyrophosphate are similar in the fetal and adult forms of the enzyme. The enzyme activity is separated into two forms by gel filtration. The enzyme from adult lung exists as a high molecular weight species, ranging in size from 5 X 10(6) to 50 X 10(6). The enzyme from fetal lung exists as a 190,000 molecular weight species and is totally dependent upon added anionic phospholipid for activity in both the forward and reverse direction. The addition of phosphatidylglycerol gives maximal activity, while phosphatidylinositol or cardiolipin produce about 60 to 70% of the maximal activity. Enzyme activation is accompanied by an aggregation of the enzyme. A sonicated preparation of phosphatidylglycerol is a more efficient activator than a preparation mixed on a Vortex mixer (KA = 30 micronM) and also converts a larger proportion of enzyme from fetal lung into a high molecular weight species. The enzyme from adult lung can be dissociated into a form in fetal lung. The dissociated species can be converted back to a high molecular weight form in the presence of phosphatidylglycerol.     

Pulmonary phosphatidic acid phosphohydrolase. Developmental patterns in rabbit lung

1. The developmental patterns of the phosphatidic acid phosphohydrolase activities in developing rabbit lung were determined using both aqueously dispersed phosphatidic acid (PAaq) and membrane-bound phosphatidic acid (PAmb) as the substrates. 2. The specific activities and the total activities of the PAmb-dependent phosphohydrolase activities in the microsomes and to a lesser extent in the homogenates increased between 26 and 30 days gestation (term 31), but decreased in the adult. The PAaq-dependent activities demonstrated a smaller increase during late gestation and a decrease in the adult. 3. There was little change in either the Paaq- or the Pamb-dependent activities in the cytosol between 25 and 30 days gestation. The total activities per g lung were increased in the adult. 4. Fractionation of adult cytosol on Bio-Gel A5m revealed PAaq-dependent activities in the void volume (Vo) (50% total), a peak with an apparent molecular mass (Mr) = 150 kdaltons (25% total) and a peak with Mr = 110 kdaltons (25% total). The PAaq-dependent peak with Mr = 150 kdaltons was not detected in the fetal cytosols. 5. Gel filtration revealed PAmb-dependent activity in the Vo (15% total), a major peak with an apparent Mr = 390 kdaltons (44% total) and minor peaks with Mr = 240 kdaltons (16% total) and Mr = 110 kdaltons (24% total). Little change was observed during development. 6. Thermal denaturation studies on he PAmb-dependent activities in the cytosols produced biphasic curves with a rapidly inactivated component and a relatively heat-stable component. The thermal denaturation profiles for the PAmb-dependent activities remained relatively unaltered throughout fetal development. The thermal denaturation profiles of the PAaq-dependent activities in the fetal cytosols were also biphasic. In contrast, the inactivation profiles of the PAaq-dependent activities in adult cytosol were monophasic.     

Development of cholinephosphotransferase in guinea pig lung mitochondria and microsomes

Development of mitochondrial and microsomal choline phosphotransferase in the fetal guinea pig lung was investigated. The activity in fetal mitochondria was more than twice of that in fetal microsomes. However, in adult lung, the enzyme was distributed mostly in microsomes. In fetal lung, both the mitochondrial and microsomal enzyme activity was greatest at approx. 81% of the total gestation period (55 days). The specific activity in the microsomal fraction then declined until term, but increased again in the 24-h newborn from 1.0 to 2.3 nmol/min per mg protein. The activity in the mitochondrial fraction declined after 61 days (2.8 nmol/min per mg) to a minimal level at term (0.6 nmol/min per mg). Although the enzyme activity decreased from day 55 (1.2 nmol/min per mg), the amount of phosphatidylcholine gradually increased between day 55 and term.     

Effects of deoxycholate and phospholipase A2 on choline and ethanolamine phosphotransferases of chicken brain microsomes.

Ethanolamine phosphotransferase (EC 2.7.8.1) and choline phosphotransferase (EC 2.7.8.2) activities were assayed in fresh microsomes from adult chicken brains with either diacylglycerols or alkylacylglycerols. Pretreatment of microsomes with 1.25 mM sodium deoxycholate, a concentration less than the critical micelle concentration, produced a slight inhibition of choline phosphotransferase activity. A deoxycholate concentration (5.0 mM) greater than the critical micelle concentration (3.0 mM) decreased the choline phosphotransferase activity by more than 70% but had no effect on ethanolamine phosphotransferase activity. Inclusion of 1.25 mM deoxycholate in the assay medium decreased choline phosphotransferase activity 35% but increased ethanolamine phosphotransferase activity 50%. The deoxycholate appeared to inactive the choline phosphotransferase. Phospholipase A2 (Vipera russelli) treatments of microsomes removed phosphoglycerides and decreased both phosphotransferase activities to a similar extent. Decreased activities are probably due to disruption of the membrane structure. Choline and ethanolamine phosphotransferase activities are apparently in different enzymes which lack specificity for the type of diglyceride. Thus, the systematic names should include 1,2-diradyl-sn-glycerol instead of 1,2-diacyl-sn-glycerol.     

Cholinephosphotransferase in rat lung. In vitro formation of dipalmitoylphosphatidylcholine and general lack of selectivity using endogenously generated diacylglycerol

Diacylglycerol was generated in vitro in rat lung microsomes by forming phosphatidic acid via sn-glycerol-3-phosphate acyltransferase followed by the hydrolysis of the phosphatidic acid by phosphatidate phosphohydrolase. Diacylglycerol concentrations of 35 to 50 nmol/mg of microsomal protein were obtained. Cholinephosphotransferase activity was determined in microsomes by measuring the conversion of endogenously generated [14C]diacylglycerol to phosphatidylcholine. Reaction rates of 14 to 16 nmol/min/mg of protein were obtained with a 30-s reaction. Diacylglycerol which was primarily dipalmitoylglycerol was produced when palmitic acid was used in the sn-glycerol-3-phosphate acyltransferase reactions. Dipalmitoylphosphatidylcholine was formed via cholinephosphotransferase from the dipalmitoylglycerol with an apparent maximal velocity of 20 nmol/min/mg of protein. When oleic acid was used instead of palmitic acid, the apparent maximal velocity for cholinephosphotransferase was 26 nmol/min/mg of protein. The apparent Km values for the two different diacylglycerol substrates were the same (28.5 nmol/mg of protein). Diacylglycerols, with different molecular species composition, were generated using a variety of fatty acids and fatty acid mixtures. The phosphatidylcholine formed from these diacylglycerols had the same molecular species profiles as the diacylglycerol used as the substrate. The relative reaction rates with the different diacylglycerols were essentially the same except when 20:4 and 22:6 fatty acids were used individually, in which case the rates were lower. We conclude that cholinephosphotransferase readily forms dipalmitoylphosphatidylcholine from endogenously generated dipalmitoylglycerol and that the cholinephosphotransferase reaction is generally nonselective for the diacylglycerol substrate.     

Properties of mannosyl- and N-acetylglucosamine-1-phosphate transferases towards dolichol phosphate in liver microsomes from pig embryos

1. The activity of mannosyl- and N-acetylglucosamine-1-phosphate transferases in microsomes from pig embryonic liver was linear to 1 min of incubation at 37 degrees C. 2. The activity of both enzymes was higher in the presence of Mg2+ as compared to Mn2+. A maximal stimulatory effect of Mn2+ was obtained at 2 mM concentration and greater concentrations of it inhibited the activities of both enzymes. 3. The activity of mannosyl transferase was found to be highest after treatment of microsomes with Nonidet P-40 while the activity of N-acetylglucosamine-1-phosphate transferase was greatest in the presence of sodium deoxycholate. 4. The Km for acceptor substrate was 1.6 x 10(-5)M in the reaction for dolichol phosphate mannose synthesis and 2.2 x 10(-5)M in the reaction for dolichol pyrophosphate N-acetylglucosamine formation. 5. The Km for GDP-mannose was 1.4 x 10(-5)M and for UDP-N-acetylglucosamine-6.2 x 10(-5)M. At saturating concentrations of donor substrates V values (pmol/min/mg) were 1330 and 150, respectively.     

Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung

Endothelial nitric oxide (NO) synthase (eNOS) produces NO, which contributes to vascular reactivity in the fetal lung. Pulmonary vasoreactivity develops during late gestation in the ovine fetal lung, during the period of rapid capillary and alveolar growth. Although eNOS expression peaks near birth in the fetal rat, lung capillary and distal air space development occur much later than in the fetal lamb. To determine whether lung eNOS expression in the lamb differs from the timing and pattern reported in the rat, we measured eNOS mRNA and protein by Northern and Western blot analyses and NOS activity by the arginine-to-citrulline conversion assay in lung tissue from fetal, newborn, and maternal sheep. Cellular localization of eNOS expression was determined by immunohistochemistry. eNOS mRNA, protein, and activity were detected in samples from all ages, and eNOS was expressed predominantly in the vascular endothelium. Lung eNOS mRNA expression increases from low levels at 70 days gestation to peak at 113 days and remains high for the rest of fetal life. Newborn eNOS mRNA expression does not change from fetal levels but is lower in the adult ewe. Lung eNOS protein expression in the fetus rises and peaks at 118 days gestation but decreases before birth. eNOS protein expression rises in the newborn period but is lower in the adult. Lung NOS activity also peaks at 118 days gestation in the fetus before falling in late gestation and remaining low in the newborn and adult. We conclude that the pattern of lung eNOS expression in the sheep differs from that in the rat and may reflect species-related differences in lung development. We speculate that the rise in fetal lung eNOS may contribute to the marked lung growth and angiogenesis that occurs during the same period of time.     

Arachidonic Acid Oxidation by Brain and Placenta Preparations from Normal and Placental Insufficient Fetal Rabbit

Cytosolic (100,000 g) fractions of fetal rabbit brain and placenta tissue convert [1-14C]arachidonic acid into several oxidation products identified with the lipoxygenase [12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE] and cyclooxygenase [prostaglandin E2 (PGE2)] pathways. Formation of 12-HETE and 15-HETE by fetal brain is time-dependent, reaching a plateau after 40 min and is linear with protein concentration. An apparent affinity constant of 0.06 mM and a Vmax of 0.1 mumol/h/g wet weight are presumably responsible for the excessive accumulation of 12-HETE and 15-HETE in comparison to PGE2 (Km = 0.5 mM). The latter is synthesized by the placenta particulate fraction but almost exclusively by the brain cytosol. Compared to brain, the activity of the placenta tissue is exceedingly higher and in addition to 12-HETE and 15-HETE there is a substantial formation of 12-L-hydroxyheptadecatrienic acid. Formation of 12-HETE and 15-HETE at 21 days is as effective as at 31 days gestation and is strongly inhibited by nordihydroguaiaretic acid (93%), BW755c (99%), and AA861 (84%) but not by indomethacin. Placenta and brain tissues of intrauterine growth retarded fetuses after ligation of placental blood vessels fail to convert arachidonic acid into other eicosanoids. Loss of enzymatic activity also observed in normal tissue after prolonged storage cannot be restored by the addition of several SH agents, ascorbate, or ferric iron.     

Characterization and distribution of a phospholipase A2 activity from adult rabbit lung

A phospholipase A2 activity was characterized in adult rabbit lung. This activity was calcium- and deoxycholate-dependent and displayed an alkaline pH optimum. Km and Vmax were 0.176 mM and 256.8 pmoles/min./mg protein respectively. The microsomal fraction displayed the highest enzymatic specific activity; the lowest activity was present in the cytosol. Yet this latter fraction accounted for the majority of the total activity. Although the specific activity was high within the lamellar body fraction this compartment contained only approximately 2% of the total activity. Phospholipase A2 activity was inhibited by bromophenacyl bromide, chlorpromazine and mepacrine in decreasing order of effectiveness. Treatment of the microsomes with increasing concentrations of NaC1 indicated that the lung phospholipase A2 activity was relatively loosely bound to the microsomal membranes and was maximally removed with salt at a concentration only slightly higher than physiological. Addition of calmodulin to the enzyme assay did not significantly alter hydrolysis of labelled phosphatidylcholine.     

Development of glycerophosphate acytransferase in guinea pig lung mitochondria and microsomes

Development of mitochondrial and microsomal glycerophosphate acyltransferase in the fetal guinea pig lung was investigated. Mitochondrial and microsomal enzyme activity gradually increased from 45 days to 55 days of gestation. The specific activity in the microsomal fraction (8.2 nmol/min per mg protein) then declined until term, but increased again in the 24-h newborn from 2.5 to 6.1 nmol/min per mg protein. Glycerophosphate acyltransferase activity in the mitochondrial fraction declined after 55 days (3.5 nmol/min per mg) to a minimum level at 60 days (1.8 nmol/min per mg), but increased again in the 24-h newborn (4.0 nmol/min per mg). The specific activity of both mitochondrial and microsomal enzyme declined after 24 h after birth until adult levels were attained. Glycerophosphate acyltransferase activity in mitochondria and microsomes from adult lung was 0.8 and 2.0 nmol/min per mg, respectively. Microsomal enzyme activity was consistently inhibited (over 95%) throughout gestation and adulthood by exposure to any one of several proteinases: trypsin, chymotrypsin, papain, bromelain, pronase and nagarse. Although mitochondrial enzyme activity was also inhibited by these proteinases, there was a continuous increase in proteinase-resistant glycerophosphate acyltransferase activity between 45 days of gestation and term. In contrast, adult mitochondrial enzyme activity was stimulated by all the proteinases studied. These results suggest that early in gestation, glycerophosphate acyltransferase lies more exposed on the cytoplasmic side of the mitochondrial outer membrane and as gestation progresses it becomes embedded into the phospholipid bilayer.     

The activity and properties of an acidic triacylglycerol lipase from adult and fetal rat lung

Triacylglycerol lipase with maximal activity at pH 5 was present in adult and fetal lung. The activity was inhibited by serum concentrations used to measure lipoprotein lipase and by 0.5 M NaCl. The activity in homogenates from fetal lung was about 40% of the activity in adult lung homogenates. The activity increased to 80% of the adult levels during the first 24–48 h following birth. Acidic triacylglycerol lipase was present in all subcellular fractions from adult lung. However, the major amount of activity appeared to be associated with lysosomes. Fetal lung contained significantly more activity in the cytosolic fraction compared to the adult. The reaction produced free fatty acids (65%), 1,2(2,3)-diacylglycerol (22%) and 2-monoacylglycerol (12%). Minimal amounts of 1,3-diacylglycerol and 1(3)-monoacylglycerol were formed. Diacylglycerol lipase and monoacylglycerol hydrolase activities at pH 5 were independently determined and both were higher than the triacylglycerol lipase activity. The subcellular distribution of diacylglycerol lipase and monoacylglycerol hydrolase differed from that of triacylglycerol lipase. Overall, the results indicated that the lung has considerable intracellular lipase activity and therefore could readily hydrolyze intracellular triacylglycerol to free fatty acids. The reaction also produced significant amounts of 1,2-diacylglycerol which suggests that triacylglycerol could be a direct source of diacylglycerol for phospholipid synthesis.     

Salinity adaptation of HCO3--dependent ATPase activity in the gills of blue crab (Callinectes sapidus)

A bicarbonate-dependent ATPase (EC 3.6.1.3) was found in microsomal preparations from blue crab gills. When the crabs were transferred to low salinity (200 mosmolal) from seawater (1000 mosmolal), the HCO3- dependent ATPase increased in all gill pairs, reaching its new steady state in 2 weeks. The greatest increase occurred in the sixth and seventh gill pairs (approx. 2.5-fold). Maximal enzyme activity was observed at an Mg2+ concentration of 2 mM and an optimal pH of 7.8. The apparent Ka for HCO3- was found to be 8.9 mM. Kinetic analysis showed that low-salinity adaptation increased the Vmax without altering the Km for ATP. When the microsomes from high-salinity crab gills were treated with detergent or assayed at different temperatures, the total enzyme activity did not reach the activity levels after adaptation to low salinity. These results suggest that the alteration of HCO3- -ATPase activity may be due to synthesis, rather than modulation of membranes or of the existing enzyme activity.     

Ontogenetic changes in levels of phosphodiesterase for adenosine 3':5'-monophosphate and glucosine 3':5'-monophosphate in the lung, brain and heart from guinea pigs.

Changes in tissue levels of the low Km phosphodiesterase for adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclc GMP) in the lung, liver, heart and brain from developing guinea pigs were studied. It was found that the contents of the soluble (cytosol) phosphodiesterase for both cyclic AMP and cyclic GMP were higher in the lung from the fetus than from the neonate and adult. The ontogenetic changes seen in the liver were qualitatively similar to thos in the lung with respect to cyclic GMP hydrolysis, while a reversed pattern of change was noted in the brain. The level of cyclic AMP phosphodiesterase was highest in the fetal heart. Throughout the fetal stage, the levels of the enzyme for cyclic GMP hydrolysis were higher than those for cyclic AMP in the lung. At or around birth, a reversal in the relative levels of the two enzymes took place; two days after birth, the level of the enzyme for cyclic AMP was 2-3times higher than thos for cyclic GMP. Kinetic analysis showed that phohphodiesterases from extracts of the lung from all developmental stages of guinea pigs had the same Km (2.6 muM) for cyclic AMP and the same Km (6.6 muM) for cyclic GMP. The relative values of V, based on assays using the same amount of enzyme protein, in decreasing order, were fetus greater than neonate greater than adult. The present findings suggest that metabolism of the two cyclic nucleotides may be closely related to developmental processes of the tissues. Moreover, the actions involving cyclic GMP may be more predominent in the fetal lung and adult brain.