Determination of calcium transport and phosphoprotein phosphatase activity in microsomes from respiratory and vascular smooth muscle

1. 1. Calcium transport into microsomal vesicles of respiratory (tracheal) smooth muscle was characterized. This calcium transport was ATP dependent and stimulated by the presence of the oxalate ion. The magnitude of transport was similar to that reported for microsomes from other types of smooth muscle.

2. 2. Bovine and rabbit, heavy and light microsomes were isolated from respiratory (tracheal) and vascualar (aortic) smooth muscle. Preincubation of these vesicles with cyclic AMP and protein kinase did not alter the transport of calcium into the vesicles. There was no evidence of phosphate incorporatio into microsomal membrane proteins. Similar results were obtained if phosphorylase b kinase replaced the combination of cyclic AMP and protein kinase during the preincubation.

3. 3. The phosphoprotein phosphatase activity of cardiac sarcoplasmic reticulum and smooth muscle microsomes was determined. The activity of this enzyme was found to be several-fold less in the cardiac sarcoplasmic reticulum than in various smooth muscle microsome preparations.

The effects of melanocortin agonists and antagonists on leptin-induced fever in rats

1. Over three experiments, separate groups of adult male Sprague–Dawley rats received intracerebroventricular (ICV) injections of either vehicle, recombinant rat leptin (1 μg), or leptin (4 μg), then two ICV injections, 30 min apart of vehicle/vehicle, leptin (4 μg)/vehicle, vehicle/α-MSH (300 ng), or leptin/α-MSH, and then vehicle/vehicle, leptin (4 μg)/vehicle, vehicle/ SHU-9119 (200 ng; a MC 3/4 receptor antagonist), or leptin/SHU-9119. Core temperatures (T_c), food intake and body weights were monitored.

2. Four microgram leptin resulted in the induction of fever, an effect blocked by injection of α-MSH. Antagonism of MC 3/4 receptors with SHU-9119 did not augment leptin-induced fever, but did block the inhibitory actions of leptin on food intake.

3. These data demonstrate the inhibitory effects of exogenous α-MSH on leptin-induced fever, but suggest that endogenous melanocortin action at MC 3/4 receptors does not tonically inhibit febrigenesis caused by leptin administration.

Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN⁻; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k_oi is much greater than k_io, it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10⁴. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, J_M and K_m of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, J_M, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the K_m for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the K_m for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high K_m and is not additive with the Na⁺-dependent flux.     

Inverse relationship of protein concentration and binding activity of alpha-galactoside receptors from sugarcane membranes

1. 1. The binding activity of purified α-galactoside receptor proteins from a number of plant species decreases when the protein concentration is increased from 2 ng/ml to 100 μg/ml.

2. 2. The apparent loss of binding activity at high protein concentrations corresponds to the formation of high molecular weight multimers.

3. 3. Raffinose and melibiose cause a ligand-dependent increase in binding activity and a corresponding decrease in the relative abundance of multimers at any given protein concentration.

4. 4. The self-inhibition of binding activity at high protein concentrations arises from a competition between ligand binding by oligomers and self-association of these oligomers into multimeric species which have little or no binding activity.

Amino acid transport and protein synthesis in energetically-stable calcium-tolerant isolated cardiac myocytes

Myocytes isolated by enzymic dispersion from adult rat ventricular tissue are shown to be energetically stable in the presence of 0.5 mM CaCl₂. ATP and ADP content and rates of lactate production are comparable with those of intact myocardial tissue and consistent with these cells being tightly coupled. Addition of 2,4-dinitrophenol precipitates rapid changes in adenine nucleotide concentrations and a 10-fold increase in lactate production. Cardiac myocytes selectively transport neutral amino acids of the A and L classes. Transport of the amino acid analogue α-aminoisobutyric acid is an active, temperature-dependent and insulin-sensitive process. The apparent K_m for α-aminoisobutyric acid transport is similar to that determined for embryonic cardiac cells. Mature myocytes incorporate labelled amino acids into cytoplasmic proteins with molecular weights ranging from 10 000 to 150 000. Newly synthesised protein is metabolically stable. The data establishes calcium-tolerant myocytes as an experimental system offering many advantages over whole hearts for short- and long-term studies of protein synthesis and catabolism.     

Chemical characterization of sialyl oligosaccharides in milk of the Japanese black bear, Ursus thibetanus japonicus

Sialyl oligosaccharides were separated from two samples of Japanese black bear milk by extraction with chloroform/methanol, gel filtration on Bio Gel P-2, ion exchange chromatography on DEAE-Sephadex A-50 and high-performance liquid chromatography (HPLC) on a TSK gel Amido-80 column. They were characterized by ¹H-NMR spectroscopy. The structures of four sialyl oligosaccharides separated from the milk were the following:

Neu5Ac(α2-3)Gal(β1-4)Glc

Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3) Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6) Gal(β1-4)Glc

Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3) Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6) Gal(β1-4)Glc

Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc