The primary structure of mitochondrial aspartate aminotransferase from pig heart: peptides obtained by cleavage with pepsin and with Staphylococcus aureus protease.

Results obtained after digestion of mitochondrial aspartate aminotransferase from pig heart with pepsin and with the protease from S. aureus are described. Peptic digestion produced a very complex mixture of peptides, which were purified and analyzed; structural information contained in these peptides covered nearly the entire molecule. Moreover, the lengths of some individual peptides and the peculiar self-overlapping found with families of peptides from adjacent regions were especially useful and interesting. Not all the possible peptides originating after digestion with S. aureus protease were isolated and examined. However, the high specificity of this protease and its usefulness for sequence studies were confirmed. In particular, the S. aureus peptides obtained were important for establishing the amidation state of glutamic acid/glutamine residues.

Structural comparison of the haemoglobin components of the armoured catfish Pterygoplichthys pardalis. Evolutionary considerations.

Amino acid analyses and peptide mapping were performed for the four main haemoglobins from the armoured catfish Pterygoplichthys pardalis; component I, which is functionally distinct from the others, is structurally unique, whereas components II, III and IV, functionally indistinguishable, are closely related in structure. Compositional difference indices are calculated for the four components and for the two major haemoglobins from the trout Salmo irideus, and the results are discussed in terms of structural relationships and evolutionary history of fish haemoglobins.

Effect of the cationic polypeptide polylysine on neutral amino acid transport in isolated brain microvessels.

The functionality of isolated brain microvessels - used as anin vitro model of the blood-brain barrier - can be influenced by interaction with cationic proteins. The various polylysines (Mr ranging from 0.9 to 180 kDa) tested affected the activity of both the Na(+)-dependent ("A") and the Na(+)-independent ("L") systems for neutral amino acid transport. Exposure to the 180 kDa polylysine caused a conspicuous inhibition of both transport systems, associated to an increased passive permeability. There was a constant, Mr-dependent, inhibition of the the L-system-mediated uptake of hydrophobic neutral amino acids. The activity of the A-system was enhanced, upon exposure to polymers larger than 22 kDa reaching its peak at 68 kDa and and declining at higher Mr values. The effect which was Na(+)-ions dependent and abolished by phloretine, could be essentially ascribed to an increased affinity of the MeAIB for its carrier (Km value decreasing from 265 to 169µM in presence of 68 kDa polylysine).

Deltorphin transport across the blood-brain barrier.

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood-brain barrier penetration rate. In vitro, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na+ ions from the incubation medium decreases the carrier affinity for deltorphins (-25%), but does not affect the Vmax value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective delta-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for delta- or mu-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that mu-, delta-, and kappa-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with L-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

Effect of inorganic phosphate on hypoxanthine transport in isolated brain microvessels.

In isolated brain microvessels, used as an in vitro model of the blood-brain barrier, the rate of hypoxanthine uptake was modulated by the presence of inorganic phosphate. A single high-capacity, low-affinity transport system was apparently active in a phosphate-free medium (Vmax = 840 pmol/mg protein/min, Km = 750/uM); in the presence of 10 mM phosphate, there was also a low-capacity, high-affinity system (Vmax = 47 pmol/mg protein/min, Km = 27/uM). The phosphate-dependent component was inactive in the absence of glucose or of Na+ ions, or upon addition of phloretine (but was scarcely affected by 2,4-dinitrophenol). This activity was apparently coupled to the intracellular phosphoribosyltransferase-catalyzed conversion of purines into the corresponding nucleotides: when inorganic phosphate was present in the suspending medium, labeled hypoxanthine was transported with higher efficiency and was readily converted to inosine monophosphate and to other related nucleotides. In the absence of phosphate ions, hypoxanthine was instead metabolized to xanthine and uric acid.

Inhibitory effect of somatostatin on neutral amino acid transport in isolated brain microvessels.

In the presence of somatostatin-14 or some of its receptorial agonists, the uptake of large neutral amino acids by isolated brain microvessels was found to be inhibited up to 50%, no other transport system being affected. Although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only uptake from the abluminal side appears to be inhibited by somatostatin. The involvement of a type-2 somatostatin receptor was suggested by assays with a series of receptor-specific somatostatin agonists, and was confirmed by the release of inhibition caused by a specific type-2 receptor antagonist. A type-2-specific mRNA was indeed shown to be present in both bovine brain microvessels ex vivo and primary cultures of endothelial cells from rat brain microvessels.