Ken C. Reed

Methodology for in vitro studies of Ca2+ transport

Abstract The difficulties inherent in measuring rates of Ca2+ transport by isolated vesicular systems are defined, together with methods for solving them. The final experimental system has been derived specifically for studies with mitochondria, but the principles involved are more widely applicable. The method involves using 45 Ca 2+ NTA (nitrilotriacetic acid) buffers to generate accurately known concentrations of 45Ca2+ and quenching of Ca2+ transport with a mixture of a high-affinity noncompetitive inhibitor (ruthenium red) and EGTA (ethanedioxy-bis-(ethylamine)-tetraacetic acid) to permit short, well-defined reaction times while simultaneously removing externally bound 45Ca2+; 45Ca2+ in the internal space is then estimated by scintillation counting of the filtered and washed suspension. This method is the most rigorous of those currently available and has the added advantage of allowing great flexibility in incubation conditions.

Sequences from a family of bovine Y-chromosomal repeats.

A 307-bp Sau3AI fragment previously cloned by deletion enrichment from the bovine Y chromosome was used to isolate a larger lambda EMBL3A genomic cattle clone. The whole 13-kb insert did not give a sex-specific pattern of hybridization to Southern blots of cattle DNA. Subclones from this phage, however, did show that this fragment had a Y-chromosomal origin. It was estimated that at least 40% of the cattle Y chromosome is composed of repeated sequences related to those within these subcloned fragments. Sequences within these subclones are male-specific or male-enriched also in sheep, goats, and deer. Comparison of cattle and sheep homologues of these sequences reveals that much greater amplification and rearrangement have occurred on the cattle Y chromosome than on the sheep Y. The apparent insertion of sequences into cattle Y-specific sequences relative to the sheep homologues suggests possible mechanisms for the evolution of the artiodactyl Y chromosome.

A low molecular weight ruthenium complex inhibitory to mitochondrial Ca2+ transport

Moore [l] and Vasington et al. [2] have demonstrated a specific inhibition of mitochondrial reactions associated with active Ca’ + transport by commercial preparations of Ruthenium Red. However, such preparations generally contain less than 20% of the pure compound [3]. Recent studies in this laboratory [4, 51, which concentrated on the mechanism of inhibition of the Ca*+ carrier by pure Ruthenium Red ([(NH& Ru-0-Ru(NH&,-0-Ru (NH&] 6+Cl& 4H2 0; [6] ), suggested that a colourless contaminant of the crude preparations has inhibitory properties similar to those of Ruthenium Red itself. Identification of the compound would be of some interest as it should provide further information on the nature of the sites involved in non-competitive inhibition of the Ca*+ carrier. A second feature, inherent in its lack of absorption in the visible spectrum, is that it would not interfere with optical measurements of mitochondrial redox components and thus would be more useful than Ruthenium Red in determining the relation between non-competitive carrier inhibition and energy transduction. This report describes preliminary attempts to isolate and identify this compound.

Occurrence of the (GATA)n sequences in vertebrate and invertebrate genomes

Clusters of the tetranucleotide GATA are found throughout the mouse genome with a major concentration on the Y chromosome. In Drosophila melanogaster, by contrast, they have a significant concentration on the X chromosome. Largely on the basis of these sex chromosomal concentrations and on their transcriptional activity in the mouse, these simple sequence tracts have been thought to be important in sex-determining and X inactivation mechanisms in both vertebrates and invertebrates. In every tested case the interpretations of the data have been difficult and sometimes the data themselves have been conflicting. We demonstrate in this paper that significant tracts of (GATA)n are totally absent from ovine and bovine genomes and point out that none of the major clusters of these repetitive sequences are near any of the sex-determining genes in D. melanogaster. We conclude therefore that (GATA)n sequences are not conserved over long evolutionary time periods as has previously been thought. Their absence from at least two mammalian genomes places severe constraints on their possible functions.

Cytoplasmic sources of NADPH for fat synthesis in rainbow trout liver: Effect of thermal acclimation on enzyme activities

Abstract 1. 1. Activity levels of the enzymes NADP malate dehydrogenase, NADP isocitrate dehydrogenase, Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NAD malate dehydrogenase and ATP citrate lyase, have been determined in cytoplasmic preparations of liver from warm and cold acclimated rainbow trout. 2. 2. The activity levels of the four NADP dehydrogenases are similar and do not change significantly following cold acclimation, suggesting that regulation of NADPH production is achieved by mechanisms other than alterations in the steady state levels of these enzymes. 3. 3. The absence of ATP citrate lyase activity may indicate that the levels are too low to detect by the assay method and the enzyme may be rate limiting in the pathway, or that a pyruvate-malate cycle is inoperative in this tissue.

An oxygen polarograph designed for undergraduate use

Abstract Details are given for the construction of a membrane-covered oxygen electrode and the electrode coupling circuit, water-jacketed reaction vessel, and water-driven magnetic stirrer, all of which are incorporated into a compact self-supporting unit. The complete unit is particularly suited to the undergraduate laboratory in view of its compactness, robustness, and ease of operation. Performance characteristics of the electrode itself equal those of commercially available Clark-type electrodes.

On the role of the adenosine diphosphate-adenosine triphosphate exchange reaction in oxidative phosphorylation: Effect of calcium

An important property of the ADP-ATP exchange reaction of oxidative phosphorylation is its susceptibility to loss of mitochondrial integrity as measured by the acceptor control ratio [l-3] . In the study of Bygrave and Lehninger [l] some indication was provided that the loss in exchange activity on loss of mitochondrial integrity was accompanied by an increase in ATPase activity. In our view this point is central in any evaluation of the mechanism of the exchange reaction in oxidative phosphorylation. We have therefore investigated this problem in greater detail by studying the response of these parameters to calcium. Our data provide substantial evidence for the view that the exchange reaction is directly related to the integrity of the isolated mitochondria and inversely related to mitochondrial ATPase. They further indicate that the likelihood of isolating this reaction from mitochondria as an ADP-ATP exchange enzyme is remote [2 ] .

Effect of temperature on the properties of cytoplasmic NADP malate dehydrogenases from liver of warm and cold acclimatized rainbow trout

Abstract 1. 1. Cytoplasmic NADP-MDH enzymes have been isolated from the livers of warm and cold acclimatized rainbow trout. 2. 2. The enzymes from the two acclimatization groups differ in the ratio of the rates of the decarboxylation to carboxylation reactions, and in their sensitivity to inhibition by NADPH at low temperature. 3. 3. It is proposed that the production of NADPH by cytoplasmic NADP-MDH in different thermal acclimation states may be controlled by changes in the occurrence of two forms displaying different and adaptive regulatory properties.