Karl B. Freeman

Identification by Site-directed Mutagenesis of Three Arginines in Uncoupling Protein That Are Essential for Nucleotide Binding and Inhibition*

Primary regulation of uncoupling protein is mediated by purine nucleotides, which bind to the protein and allosterically inhibit fatty acid-induced proton transport. To gain increased understanding of nucleotide regulation, we evaluated the role of basic amino acid residues using site-directed mutagenesis. Mutant and wild-type proteins were expressed in yeast, purified, and reconstituted into liposomes. We studied nucleotide binding as well as inhibition of fatty acid-induced proton transport in wild-type and six mutant uncoupling proteins. None of the mutations interfered with proton transport. Two lysine mutants and a histidine mutant had no effect on nucleotide binding or inhibition. Arg83 and Arg182 mutants completely lost both the ability to bind nucleotides and nucleotide inhibition. Surprisingly, the Arg276 mutant exhibited normal nucleotide binding, but completely lost nucleotide inhibition. To account for this dissociation between binding and inhibition, we propose a three-stage binding-conformational change model of nucleotide regulation of uncoupling protein. We have now identified three nucleotides by site-directed mutagenesis that are essential for nucleotide interaction with uncoupling protein.

Loss of brown adipose tissue uncoupling protein mRNA on deacclimation of cold-exposed rats

The effect of environmental temperature on the level of uncoupling protein mRNA from rat brown adipose tissue was examined using a cDNA probe. A 4.4 fold increase in the mRNA level was observed after 1 day exposure of rats to 6 degrees C, which was followed by a slow loss with longer times of exposure. When rats were returned to a thermoneutral environment, there was a dramatic loss of uncoupling protein mRNA within 1 day. Comparison wih poly(A)+ RNA levels suggest that the response to temperature is specific for uncoupling protein mRNA.

Site of biosynthesis of mammalian cytochrome c oxidase subunits

In Saccharomyces cerevisiae [l-3] and in Neurospora crassa [ 1,4] it has been shown that the three largest subunits of cytochrome c oxidase (EC 1.9.3.1) are synthesized in mitochondria, while the remaining subunits are synthesized on cytosolic ribosomes. In Locusta migratora, at least one of the subunits of insect cytochrome c oxidase is synthesized in mitochondria [S] . Previous work with mammalian cells has suggested a mitochondrial origin for at least some of the subunits of cytochrome c oxidase because formation of the enzyme is inhibited by chloramphenicol or its sulfamoyl analog, Tevenel [ 1,6,7] . The present work provides immunological evidence that at least two of the subunits of mammalian cytochrome c oxidase are synthesized in mitochondria.

Synthesis in vitro of rat brown adipose tissue 32000 Mr protein

The synthesis of the mitochondrial inner membrane 32 000 M r protein from rat brown adipose tissue was examined. Polysomes from the tissue were translated in a reticulocyte lysate protein‐synthesizing system and newly‐synthesized protein isolated with a monospecific antibody against the 32 000 M r protein. The newly‐synthesized protein had the same relative molecular mass as the mature protein. It was taken up by mitochondria isolated from Chinese hamster ovary cells into a form resistant to trypsin.

A putative precursor of rat liver mitochondrial malate dehydrogenase.

Mitochondria have their own genetic system but the majority of mitochondrial proteins are coded by nuclear DNA, synthesized on cytosolic ribosomes and imported into mitochondria [ 1,2]. Little is known of the mechanism of import [3-61. There is some evidence that the mature form or subunits of the mitochondrial matrix proteins aspartate aminotransferase [6-91 and malate dehydrogenase (EC 1 .l .1.37) [6,1 O-l 21 can be taken up into the matrix of isolated mitochondria. However, a number of mitochondrial matrix proteins including aspartate aminotransferase [ 131 are synthesized in precursor form [3,5]. Here, we report a putative precursor or rat liver mitochondrial malate dehydrogenase which is synthesized in a reticulocyte lysate primed with either free polysomes or total RNA from rat liver.

Conservation of Genes Coding for Proteins Synthesized in Human Mitochondria

Proteins synthesized in mitochondria of 27 different human cell lines, identified by labeling with [35S]methionine in the presence of cycloheximide, have been enumerated and their electrophoretic mobilities determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Twelve bands were observed in all cell lines. In 24 cell lines, the electrophoretic mobilities of the proteins were the same regardless of race, sex, tissue of origin, cell type, viral transformation, or premature biological aging syndromes. The patterns obtained for the remaining cell lines, HeLa, KB, and Hep-2 were identical. These cell lines showed one protein component that was absent in the 24 others, and lacked a component present in these cell lines. Since it has been previously asserted that KB and Hep-2 are HeLa cells, the data indicate that one basic pattern exists in human cells with a variant of unknown origin occurring in HeLa cells.

Immunological detection of cDNA clones encoding the uncoupling protein of brown adipose tissue: Evidence for an antigenic determinant within the C-terminal eleven amino acids

Poly(A)+ RNA was isolated from brown adipose tissue of cold acclimated rats and a fraction enriched for uncoupling protein mRNA was used to generate a cDNA library in pBR 322, Immunological screening of 1,500 colonies with an affinity-purified antiserum against the uncoupling protein yielded five positive clones, pUCPratl–5. Clone pUCPrat2 encoded the C-terminal 54 amino acids of rat uncoupling protein and exhibited 90% amino acid homology with the hamster protein. Clones pUCPrat3–5 encoded only the C-terminal 11 amino acids suggesting that an antigenic determinant lies within this sequence.

Multiple species of methionyl-tRNA from mouse liver mitochondria

Methionyl tRNA acylated within mitochondria isolated from mouse liver, has been resolved into four species by RPC-5 chromatography. All four elute prior to the three cytoplasmic methionyl tRNA species. Of the four species, two are formylated. These results suggest that iso-accepting species of met-tRNAmet exist in mouse liver mitochondria.

Decrease in hepatic cytochrome P-450 and catalase following allylisopropylacetamide: The effect of concomitant hemin administration

Abstract It is known that administration of allylisopropylacetamide (AIA) to rats increases δ-aminolevulinic acid synthetase (ALA-S), urine ascorbic acid excretion and decreases hepatic hemoproteins cytochrome P-450 and catalase. Hemin has been shown to inhibit induction of ALA-S by AIA. To investigate further the regulatory role of hemin, AIA was administered to rats over a 5 day period with and without hemin. Hemin did not prevent the decrease in P-450 or catalase caused by AIA but partially inhibited induction of ALA-S and the increase in ascorbic acid excretion. It is unlikely that the hemin is replacing endogenous heme in hemoprotein synthesis and we conclude that the role of hemin in suppressing induction phenomena that follow AIA treatment reflects a regulatory function not related to a role in hemoprotein synthesis.

The synthesis of ribosomal-type rna by isolated rat liver mitochondria

Abstract Isolated rat liver mitochondria have been shown to synthesize RNA which has an electrophoretic mobility of “21S” and “12S” on agarose-acrylamide gels. The synthesis of these RNA species was inhibited by ethidium bromide. From these observations it is suggested that mammalian mitochondrial DNA codes for mitochondrial ribosomal-type RNA.