The oxidation of oxyhaemoglobin by glyceraldehyde and other simple monosaccharides.

Glyceraldehyde and other simple monosaccharides oxidize oxyhaemoglobin to methaemoglobin in phosphate buffer at pH 7.4 and 37 degrees C, with the concomitant production of H2O2 and an alpha-oxo aldehyde derivative of the monosaccharide. Simple monosaccharides also reduce methaemoglobin to ferrohaemichromes (non-intact haemoglobin) at pH 7.4 and 37 degrees C. Carbonmonoxyhaemoglobin is unreactive towards oxidation by autoxidizing glyceraldehyde. Free-radical production from autoxidizing monosaccharides with haemoglobins was observed by the e.s.r. technique of spin trapping with the spin trap 5,5-dimethyl-l-pyrroline N-oxide. Hydroxyl and l-hydroxyalkyl radical production observed from monosaccharide autoxidation was quenched in the presence of oxyhaemoglobin and methaemoglobin. The haemoglobins appear to quench the free radicals by reaction with the free radicals and/or the ene-diol precursor of the free radical.     

Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.     

Inhibition of enzymes by metal ion-chelating reagents. The action of copper-chelating reagents on diamine oxidase

1. The inhibition of diamine oxidase has been studied by using the following copper-chelating reagents: 1,10-phenanthroline; 2,2'-bipyridyl; 8-hydroxyquinoline (oxine); diethyldithiocarbamate and dithio-oxamide (rubeanic acid). 2. Addition of chelating reagent caused a rapid inhibition of enzyme to a degree dependent solely on the final inhibitor concentration. Addition of substrate gave linear initial rates of reaction showing that under these conditions the inhibition was not being rapidly reversed. 3. The inhibition has been investigated by using new graphical methods and has been found in all cases to involve the chelating agents completely removing two Cu(2+) ions from the enzyme. An alternative possibility, involving ligand substitution, was eliminated. 4. A value of K=8.0x10(-33)m(-2) has been found for the enzyme in equilibrium with 2 Cu(2+) ions (i.e. beta(2), the stability constant for diamine oxidase/two Cu(2+), is 32.1).     

Partial sequence homologies between cytoskeletal proteins,c-myc,Rous sarcoma virus and adenovirus proteins,transducin, and β- and γ-crystallins

Computer based sequence comparisons indicate partial sequence homology between human c-myc, Rous sarcoma virus, adenovirus 7, and simian sarcoma virus proteins and the cytoskeletal proteins desmin, keratin and vimentin. In addition, sections of the oncogene proteins showed partial but significant homology to and subunits of transducin, -II and -BP crystallins showed partial but significant homology to the cytoskeletal proteins keratin, vimentin, desmin, and -tubulin, and to adenovirus 7 and simian sarcoma virus transforming gene proteins. -BP crystallin showed partial but significant homology to Rous sarcoma virus protein, and to and y subunits of transducin. Both crystallins showed partial sequence homology to the GTP-binding protein elongation factor TU fromEscherichia coli . These sequence homologies suggest a link between the mechanisms of normal lens cell differentiation, involving modifications to the cytoskeleton and subsequent changes to the pattern of protein synthesis, and mechanisms of neoplastic transformation. Furthermore the transducin-like region on -crystallin may be important for its interaction with lens membranes and the maintenance of short-range order for lens transparency.     

Structure and organization of the bovine beta-globin genes

Genomic clones spanning the entire cow beta-globin gene locus have been isolated and characterized. These clones demonstrate that the linkage of embryonic-like (epsilon) genes and pseudogenes (psi) to the previously described fetal (gamma) and adult (beta) genes is as follows: 5'-epsilon 3-epsilon 4-psi 3-beta-epsilon 1-epsilon 2-psi 1- psi 2-gamma-3'. Present data indicate that, like that of the goat, the fetal and adult genes arose via block duplication of an ancestral four- gene set: epsilon-epsilon-psi-beta. This duplication event preceded the divergence of cows and goats, which occurred greater than or equal to 18-20 Myr ago. However, cows do not have the additional four-gene block containing a preadult/stress globin gene (beta C). Furthermore, the cow fetal cluster contains an extra beta-like pseudogene, which apparently arose by a small-scale duplication. The fixation of this duplication may indicate a possible evolutionary role for pseudogenes.