Dietary lithium during development: Changes in amino acid levels,ionic content,and [3H]spiperone binding in the brain of rats

We found that chronic lithium diet affects the sensitivity of neuroleptic receptors and the content of amino acids in the brain, and that the changes in adult animals differ from those in young rats. Pregnant rats were kept on lithium diet (pellets with 0.21% Li2CO3 and 0.21% NaCl) during the gestation period and the offspring were kept on lithium for six weeks after delivery. Control rats were kept on normal diet under identical conditions. In corpus striatum and cerebral cortex of lithium-treated young rats a reduction in apparent dissociation constant and no change in (3H)spiperone total binding sites were found, suggesting a sensitization of the neuroleptic receptor; this result was unlike that obtained with adult lithium-treated rats, where the total number of binding sites was decreased. The lithium content of brain was very high (2.32 meq/kg of wet weight), whereas in the serum only 0.75 meq/l was recorded. K+ and Na+ levels increased by 20% and 9% respectively in the brain and remained at normal levels in the serum. Analysis of free amino acids in the cerebral cortex, midbrain, and cerebellum showed increases in GABA and glycine levels in all three regions, a significant increase in taurine in midbrain, and an increase in lysine in cerebral cortex and cerebellum. The results indicate that the effect of chronic dietary lithium given during pregnancy on the neuroleptic receptor in young rats is different from that in adult animals. It produces an increase in the number of the neuroleptic receptor sites instead of the decline in the number of binding sites found in adult rats. It remains to be established whether this effect is related more to the age of the animal tested or to the stage of development of the CNS at which the lithium was administered.     

Enkephalin-binding systems in human plasma

Three amino acid-containing fractions present in human plasma are shown to bind both leu and met-enkephalin: serum albumin and two species of a much lower molecular weight, in all likelihood polypeptides. The amount of enkephalin associated with serum albumin seems comparatively smaller than that associated with the two low molecular weight systems. These systems jointly are apparently capable of binding a significant part of the circulating enkephalins. The possibility is suggested that the interactions described may play a role in maintaining the integrity of circulating enkephalins.     

Heterogeneity in 5′-nucleotidase activity of mouse peritoneal macrophages

Summary After stimulation of the mouse peritoneal cavity with newborn calf serum (NBCS), four types of monocyte and macrophage were distinguished on the basis of peroxidase (PO) patterns. Cytochemically, these cells showed strong heterogeneity in 5-nucleotidase (5N) activity. Monocytes and monocyte-derived macrophages with PO activity in granules lacked 5N activity. Resident macrophages (with PO activity in RER and nuclear envelope) generally had significant 5N activity on the plasma membrane, the pattern showing close correlation with the biochemical findings. The group of PO-negative macrophages comprised both 5N-negative and 5N-positive cells. These findings suggest two possibilities, i.e., that monocytes (5N-)transform via PO-negative cells (5N-/+) into resident macrophages (5N+), or that the monocytes and monocyte-derived macrophages and the resident macrophages represent separate lineages. The fourth type of macrophage, the exudate-resident cell (with PO activity both in granules and in the RER and nuclear envelope), occurred only in low numbers and very late after NBCS stimulation, and is therefore considered not to be a transitional cell between monocytes and resident macrophages.     

Determination of proton dissociation constants by ion-exchange high-performance liquid chromatography

The common methods to determine dissociation constants of solutes, e.g., uv spectrophotometry, potentiometry, and conductimetry, are accurate but require at least 1 nmol of compound. High-performance liquid chromatography (HPLC) allows 1 pmol of a uv-absorbing compound to be detected. By adjusting the polarity of the mobile phase, reverse and normalphase properties of an ion-exchanger can be minimized, resulting in a high correlation between charge and retardation of the solute. Thus, the degree of ionization of several compounds was monitored in mobile-phase compositions of different pH values using cation exchange. The pK values of several pterin derivatives corresponded to those obtained by other methods. In addition, pK values of two unidentified pterin derivatives were determined, using only 20 pmol of each.     

Continuous titration of difference absorption upon binding of 4-methylumbelliferyl glycosides to concanavalin A and peanut agglutinin

A continuous titration of absorption differences is described. Equal volumes of the titration fluid are dispensed from two micrometer-driven Hamilton gas-tight syringes into two 1 × 1 × 4.5-cm cuvettes. These are placed in the reference and sample beam. Each cuvette stopper is equipped with a capillary inlet connected to a syringe and with a minimotor for continuous stirring. Details of the stirring device are given. The delivered volumes of titration fluid are sufficiently reproducible to allow titration of absorption differences as a function of chromophore concentration. The usefulness of this approach is tested with the binding of 4-methylumbelliferyl α-d-mannopyranoside and concanavalin A as a well-characterized system. It is applied to the binding of similarly labeled anti-t disaccharide with the lectin from peanuts. With both lectins, the change in molecular extinction coefficient of the ligand and the association constant, valid for the entire protein saturation range, were obtained. The results are identical to those from other methods, including equilibrium dialysis.     

Comparison of detergent-solubilized and membrane-bound forms of kidney (Na + K)-ATPase

The detergent solubilization of dog kidney (Na + K)-ATPase has been investigated. The nonionic detergents, Brij 58, C₁₂E₈, and Lubrol WX were tested for their ability to produce active, soluble enzyme. Lubrol WX gave the best results. Enzyme so treated is found in the supernatant fraction after centrifugation at 100,000g for 1 h. It has the same or slightly greater specific activity, the same subunit composition as judged by SDS-gel electrophoresis, and very similar kinetic parameters with respect to sodium, potassium, ATP, pNPP, and ouabain as the membrane-bound enzyme. The Lubrol-treated enzyme is stable for at least 5 days at 4 °C. The phospholipid content of the Lubrol-treated enzyme is decreased, as might be expected, by about 50%. Limited tryptic proteolysis and fluorescence changes seen after modification with FITC indicate that the solubilized (Na + K)-ATPase undergoes the same conformational transitions as the membrane enzyme. Our results indicate that kidney enzyme solubilized as described here is nondenatured and fully active, and therefore a valuable preparation for spectroscopic and other approaches for study of this enzyme.     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.     

Isolation and characterization of a fragment of rat thyroglobulin gene

A rat genomic library was screened for thyroglobulin gene clones with recombinant plasmids containing rat thyroglobulin complementary DNA inserts. Two identical recombinant phages were found. A map of the inserted genomic sequence established by restriction and blotting experiments, and electronic microscopy revealed that this fraction of the gene was extensively split. Exons were ≤ 200 base pair long while the introns represented 93% of the insert. A fragment subcloned in plasmid pBR 322 was shown to contain repetitive sequences when used in Southern blot experiments with rat total genomic DNA.