Measurement by HPLC of Desferrioxamine-Available Iron in Rabbit Kidneys to Assess the Effect of Ischaemia on the Distribution of Iron Within the Total Pool

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10µMolar region could be accurately and reproducibly measured using this technique.

The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.     

Identification of a new subpopulation of triad junctions isolated from skeletal muscle; Morphological correlations with intact muscle

Summary It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca²⁺-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, weak, and breakage resistant, strong, triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Structure of a ricin mutant showing rescue of activity by a noncatalytic residue.

Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gln reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins: Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.     

Nonserine esterases from rat liver cytosol

An effort to identify the major general esterases of rat liver cytosol that are insensitive to the serine esterase inhibitor paraoxon (diethyl 4-nitrophenyl phosphate) has led to the isolation of a dozen enzymes. Four of these are electrophoretically homogeneous. Although purified on the basis of their hydrolytic activity toward 4-nitrophenyl acetate, each of the enzymes has a very broad and overlapping substrate specificity for aromatic esters. Thiol esters serve as substrates but, within the limits of the methods used, amides are not hydrolyzed.     

The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.