High affinity uptake of L-glutamate and γ-aminobutyric acid inDrosophila melanogaster

Preparations having properties resembling those of synaptosomes have been isolated from whole fly homogenates ofDrosophila melanogaster using ficoll gradient floatation technique. These have been characterized by marker enzymes and electron microscopy and binding of muscarinic antagenist³H Quinuclidinyl benzilate. An uptake system for neurotransmitter, ã-Aminobutyric acid has been demonstrated in these preparations. A high affinity uptake system for L-glutamate has also been studied in these subcellular fractions. This uptake of glutamate is transport into an osmotically sensitive compartment and not due to binding of glutamate to membrane components. The transport of glutamate has an obligatory requirements for either sodium or potassium ions. Kinetic experiments show that two transport systems, withK _m values 0.33 X 10^-6M and 2.0 X 10^-6M, respectively, function in the accumulation of glutamate. ATP stimulates lower affinity transport of glutamate. Inhibition of glutamate uptake by L-aspartate but not by phenylalanine and tyrosine indicates that a common carrier mediates the transport of both glutamate and aspartate. β-N-oxalyl-L-β β-diamino propionic acid and kainic acid, both inhibitors of glutamate transport in mammalian brain preparations, strongly inhibited transport of glutamate inDrosophila preparations Comparison with uptake of ã-aminobutyric acid and glutamate in isolated larval brain is presented to show that the synaptosome-like preparations we have isolated are rich in central nervous system derived structures, and presynaptic endings from neuromuscular junctions.     

(2')3',5'-Bisphosphate nucleotidase

(2')3',5'-Bisphosphate nucleotidase has been prepared in electrophoretically homogeneous form from guinea pig liver. The enzyme catalyzes the hydrolysis of the 2'- or 3'-phosphate from the appropriate nucleoside 2',5'- and 3',5'-bisphosphates and is active with 3'-phosphoadenosine 5'-phosphosulfate and with coenzyme A but not with ATP. The 40,000-dalton protein is a monomer that requires Mg2+ for activity.     

Chromium(III)-insulin derivatives and their implication in glucose metabolism

Insulin has been reacted with five chromium(III) complexes that are capable of relatively facile substitution of aquo ligands. The new Cr(III) insulin derivatives have been characterized by means of electronic and infrared spectra, and evidence for major changes in the protein structure, including the state of aggregation, has been presented. Supporting evidence for the arguments favoring the beneficiary role of chromium(III) in glucose metabolism has been obtained using in vivo studies, and it has been shown that insulin derived with Cr(salen) (H2O)2+ is capable of reversing the blood sugar, serum cholesterol, and phospholipids levels to those of normal rats. The results emphasize the dependence of biopotency on the structure of Cr(III) complexes used for derivation of insulin and discount the postulates that Cr(III) serves to assemble insulin and receptor units through metal-sulphur bonding. The influence of Cr(III) on the structural stability and state of aggregation of insulin and their possible role in glucose metabolism is discussed.     

Chymotrypsin-catalyzed hydrolysis of chromium (III) derivatives of insulin: evidence for stabilization of the protein through interactions with metal ions

We investigated the chymotrypsin-promoted hydrolysis of a series of chromium(III)-insulin complexes containing chelating or macrocyclic ligands. It has been shown that Cr(III) stabilizes insulin against the chymotrypsin-promoted hydrolysis of the protein. The molecular weights of Cr(III) containing peptides have been estimated to be of the order of 2,700-3,700 daltons. The Cr(III) containing peptides are richer in glutamic acid than the intact insulin and are devoid of any isoleucine. High molecular weights and the observed glutamic acid/histidine ratios in Cr(III) containing peptides have been rationalized in terms of Cr(III) being associated with insulin aggregates rather than the monomer of the protein. The chymotrypsin hydrolysis of Cr(III) insulin derivatives is influenced markedly by the nature, charge, and type of Cr(III) complex with which the protein has been reacted. Arguments have been advanced that chymotrypsin-promoted hydrolysis of insulin Cr(III) derivatives does not lead to cleavages at or near every tyrosine residue.     

Characteristics of tripeptide transport in human jejunal brush-border membrane vesicles

These studies are aimed at characterizing the transport of the tripeptide, glycylglycyl-L-proline (GlyGlyPro) across human jejunal brush-border membrane vesicles. GlyGlyPro (0.65 mM) was hydrolyzed by brush-border membrane vesicles with the extent of hydrolysis per mg protein being 23% at 0.5 min, 57% at 1 min and complete hydrolysis at 60 min. Treatment of the membrane vesicles with gel-complexed papain (to remove membrane peptidases) resulted in minimal hydrolysis of GlyGlyPro up to 10 min of incubation. Measurement of GlyGlyPro influx with papain-treated vesicles in the presence of increasing medium osmolarity showed that uptake occurred into an osmotically reactive intravesicular space. Transport of GlyGlyPro with normal and papain-treated membrane vesicles was similar in the presence of an inward Na+ or K+ gradient. No overshoot phenomenon was observed in the presence of an inward proton gradient (extravesicular pH 5.5; intravesicular pH 7.5). An interior negative membrane potential induced by a K+ diffusion potential in the presence of valinomycin stimulated the uptake of the peptide. The effect of increasing concentrations on initial rates of GlyGlyPro uptake revealed the presence of a saturable component as well as a diffusional component. Preloading the membrane vesicles with 20 mM glycylsarcosylsarcosine stimulated uptake by 4-fold. Uptake of GlyGlyPro was inhibited greater than 50% by dipeptides and tripeptides and less than 15% by free amino acids. These results indicate that GlyGlyPro uptake in jejunal brush-border membrane vesicles is not energized by a Na+ or proton gradient and that transport occurs by carrier-mediated and diffusional processes.     

Post-embedding methods for immunolocalization of elastin and related components in tissues

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.     

Formation of wheat protein bodies: Involvement of the Golgi apparatus in gliadin transport

Developing wheat (Triticum aestivum L.) endosperm was examined using ultrathin sections prepared from tissues harvested at 5, 9, 16 and 25 d after flowering. Protein bodies were evident by 9 d and displayed a variety of membranous structures and inclusions. The Golgi apparatus was a prominent organelle at all stages, and by 9 d was associated with small electron-dense inclusions. By immunocytochemical techniques, gliadin (wheat prolamine) was localized within these vesicles and in homogeneous regions of protein bodies, but not in the lumen of the rough endoplasmic reticulum. The protein bodies appear to enlarge by fusion of smaller protein bodies resulting in larger, irregular-shaped organelles. The affinity of the Golgi-derived vesicles for gliadin-specific probes during the period of maximal storage-protein synthesis and deposition indicates that this organelle includes the bulk, if not all, of the gliadin produced. The involvement of the Golgi apparatus in the packaging of gliadins into protein bodies indicates a pathway which differs from the mode of prolamine deposition in other cereals such as maize, rice and sorghum, and resembles the mechanism employed for the storage of rice glutelin and legume globulins.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - DAF days after flowering