Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding

[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.     

Outdoor mass culture of Spirulina maxima in sea-water

Summary The results of a whole year experiment on the outdoor mass culture of Spirulina maxima strain 4Mx on fertilized sea-water are reported. Carbonate and phosphate precipitation in the sea-water media was prevented by maintaining a low concentration of phosphate and by controlling the pH in the range of 8.0–8.3. The mean annual yield of biomass on sea-water plus urea as nitrogen source was 7.35 g (dry weight) m^-2· day^-1, a value slightly lower than that obtained on the standard bicarbonate medium (8.14 g · m^-2 · day^-1). On sea-water plus nitrate the yield was only 5.2 g·m^-2·day^-1. The nitrogen content of the biomass was higher in summer and lower in winter. The seasonal effect was more evident when nitrate was the nitrogen source.     

Inactivation of rat liver HMG-CoA reductase phosphatases by polycarboxylic acids

Incubation of the four purified HMG-CoA reductase phosphatases with the sodium salts of eleven polycarboxylic acids at concentrations of 40 mM, inactivated the enzymes to different degrees depending on the structure of the carboxylic acids. Maleate, malonate, oxalate, citrate, and hydroxymethylglutarate produced full inactivation at the concentration tested. When the four phosphatases were incubated with these acids, a concentration-dependent inactivation was observed. Fumarate, the trans isomer of maleate, produced little inactivation of the four phosphatases. Mevalonate did not inactivate at all. A relationship between those concentrations of acid that produced a 50% inactivation and the logarithm of the stability constant of Mg2+ or Mn2+ salts of polycarboxylic acids was observed. When reductase phosphatases were incubated with mixtures of polycarboxylic sodium salts and Mg2+ or Mn2+, an increase in the molar ratio divalent cation/carboxylic acid determined an increase in the four reductase phosphatase activities. The inactivating effect of citrate was on the phosphatases (high and low forms) and not on the substrates (HMG-CoA reductase, phosphorylase, and glycogen synthase). Reactivation of the citrate-inactivated phosphatases by Mn2+ and Mg2+ depended on the phosphorylated substrates, Mn2+ being the better activator. It is concluded that HMG-CoA reductase phosphatases are metalloenzymes.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Succulence and CAM relationships in Aeonium genus

The CAM has been tested in six species of the Aeonium genus by studying the diurnal fluctuation of organic acids, pH and night fixation of CO₂. The existence of a mesophyll structure able to support this metabolism has been shown as well as a congruent periodicity in the pool of cell starch. We have calculated the S, ES and Sm indices in the six species. A series of regression equations of different grades and types were calculated and shown to have correlation coefficients statistically significant. This allows us to confirm the suitability of the Sm index as a rapid test to establish the CAM as postulated by former authors.     

Interstitial fluid pressure changes in pregnant rats

In pregnant rats significant interstitial fluid pressure changes could be detected by means of capsules chronically implanted into the subcutaneous tissue. The capsular pressure increased significantly from a control value of -4.3 +/- 0.5 mmHg to -0.7 +/- 0.5 mmHg during the first period of pregnancy. Immediately before parturition the capsular pressure returned to the control level. During lactation the pressure rose as high as + 0.5 +/- 0.9 mmHg. After lactation the pressure returned again to the control value. By determining the extracellular fluid and plasma volume, as well as protein concentration in plasma and capsular fluid, the hydrostatic and colloid osmotic forces operating in the extracellular space could be analysed. It has been concluded that the observed capsular pressure changes during pregnancy are not solely of volumetric or colloid osmotic origin.     

Protein kinase activity of purified components of the chicken oviduct progesterone receptor

Preparations of the 90K and 110K components of the chick oviduct progesterone receptor (PR) purified to near homogeneity were tested for protein kinase activity. The 90K component was shown to incorporate radioactive phosphate from [γ-³²P]-ATP in the presence of Ca²⁺ but not of Mg²⁺ ions, while the 110K component was phosphorylated in the presence of Mg²⁺, but not of Ca²⁺. The enzymatic activity of the 90K polypeptide appeared selective, since added proteins (histones) did not become phosphorylated. However, all proteins present in the 110K preparations were phosphorylated in the presence of Mg²⁺. These data suggest that components of the chick oviduct PR display protein kinase activity.     

Modulation of endogenous prostaglandins by thymosin-α1 in lymphocytes

The effects of thymosin-α₁ on the stimulation of specific release of prostaglandin E₂ (PGE₂) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-α₁ in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE₂ between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-α₁ at certain concentrations was able to stimulate PGE₂ release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE₂ after incubation with α₁ in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentration of α₁ was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of α₁ after administration of this synthetic molecule show a clear dissociation. A maximum peak of α₁ activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by α₁ of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of α₁ requires prostaglandin biosynthesis.     

Estimation of intracellular fluid volume of L cells

Dilution of ¹⁴C-sucrose solution by intracellular fluid released as a result of ultracentrifugation was used to estimate the intracellular fluid volume of L cells. Consistent relationships to total cell volume as estimated by use of an electronic particle counter were obtained. Expressed as a percentage of total cell volume, the mean value plus or minus the S.D. for 6 experiments was 72.8 ± 0.9.