Directional electron transfer in ruthenium-modified horse heart cytochrome c

Cytochrome c can be modified by [(NH3)5RuII/III-] specifically at the imidazole moiety of histidine 33, and we have recently discussed the thermodynamics and kinetics of electron transfer within this modified protein. X-ray crystal structures of the oxidized and reduced forms of tuna cytochrome c indicate that the separation between the haem group of cytochrome c and the ruthenium label is 12-16 A. Internal electron transfer from the [(NH3)5RuII-] centre to the Fe(III) haem centre occurs with a rate constant k congruent to 53 s-1 (25 degrees C) (delta H = 3.5 kcal mol-1, delta S = -39 EU), as measured by pulse radiolysis. The measured unimolecular rate constant, k congruent to 53 s-1, is on the same timescale as a number of conformational changes that occur within the cytochrome c molecule. These results raise the question of whether electron transfer or protein conformational change is the rate limiting step in this process. We describe here an experiment that probes this intramolecular electron transfer step further. It involves reversing the direction of electron transfer by changing the redox potential of the ruthenium label. Electron transfer in the new ruthenium-cytochrome c derivative described here is from haem(II) to the Ru(III) label, whereas in (NH3)5Ru-cytochrome c the electron transfer is from Ru(II) to haem(III). Intramolecular electron transfer from haem(II) to Ru(III) in the new ruthenium-cytochrome c described here proceeds much slower (greater than 10(5) times) than the electron transfer from Ru(II) to haem(III) in the (NH3)5Ru-cytochrome c. We therefore conclude that electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality.     

Structure of pre-pro-von Willebrand factor and its expression in heterologous cells

Von Willebrand factor (vWF), a multifunctional haemostatic glycoprotein derived from endothelial cells and megakaryocytes, mediates platelet adhesion to injured subendothelium and binds coagulation factor VIII in the circulation. Native vWF is a disulphide-bonded homopolymer; the monomeric subunits, of apparent relative molecular mass (Mr) 220,000 (220K) are derived from an intracellular precursor estimated at 260-275K. Multimer assembly is preceded by the formation of dimers, linked near their C-termini, which then assemble into filamentous polymers. The importance of the removal of the large vWF pro-polypeptide during multimer assembly, and whether this or other stages of the complex post-translational processing require components specific to endothelial cells or megakaryocytes, is unknown. Here we report an analysis of the complete sequence of pre-pro-vWF and expression of the molecule in heterologous cells. The vWF precursor is composed of several repeated subdomains. When expressed in COS and CHO cells, it is cleaved and assembled into biologically active high relative molecular mass disulphide bonded multimers. This suggests that the information for assembly of this complex molecule resides largely within its primary structure.     

Long-range colour-generating interactions across the retina

The existence of colour-generating interactions across the corpus callosum has recently been suggested from observations with a 'split-brain' patient, thus indicating long-range colour computations at the cortical level. Observations on induced colours described here suggest long-range colour computations at the retinal level. If a white surface surrounded by a particular colour is fixated for some time, the resulting after-image has two colours: the surround appears in complementary colour, whereas the white centre takes on the colour of the surround. The question of whether such colour induction is located in the retina or more centrally was tested in a brain-injured patient with hemianopia. It could be demonstrated that areas of the visual field that are no longer represented in the geniculo-striatal pathway still contribute to colour induction, suggesting that colour induction is a retinal phenomenon.     

Ratio between large and small carboxy-terminal molecular forms of cholecystokinin in brains of different species

The ratio between large and small carboxy-terminal forms of cholecystokinin in brain extracts from man, pig, dog, rat, chicken, frog and trout was determined by two sequence-specific radioimmunoassays. It was found that the relative amounts of large forms of cholecystokinin; are higher in mammalian brain than in brains of lower species.     

Immunoquantitation of some cytochrome P-450 isozymes in liver microsomes from streptozotocin-diabetic rats

Streptozotocin-diabetes in rats leads to a decrease of cytochrome P-450 UT-A (the major form in control rats) and an increase of cytochrome P-450 PB-B (the major one induced by phenobarbital treatment) in liver microsomes. The increased benzphetamine-N-demethylase activity can be related to the induction of cytochrome P-450 PB-B.