PubMed Health⌕ Search

Biomedical subjects

K B Freeman

Publications and source records attributed to K B Freeman.

At least 37 records · Page 2Linked to original sources
PubMed1986

Mitochondrial malate dehydrogenase and its precursor have different conformations.

Antiserum prepared against the denatured form of mammalian malate dehydrogenase was found to immunoprecipitate the denatured but not the native form of the mature enzyme. In contrast, the antiserum immunoprecipitated the enzyme's precursor, synthesized in a rabbit reticulocyte lysate, either before or after denaturation. The mature form of the enzyme but not the precursor bound to an affinity column of 5'-AMP-Sepharose. These results indicate that the mature and precursor forms of malate dehydrogenase have different conformations.

Adenosine Monophosphate↗
PubMed1986

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein.

Adipose Tissue, Brown↗
PubMed1986

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, pUCPrat1-5. 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.

Adipose Tissue↗
PubMed1986

Experimental approaches to the study of the biogenesis of mammalian mitochondrial proteins.

Mitochondrial proteins are synthesized in mitochondria and on cytosolic ribosomes. Several approaches used to establish the site of synthesis and the identity of mitochondrially synthesized proteins are described. These include the specific inhibition of mitochondrial translation by inhibitors or mutation and the specific elimination of cytosolic translation either by using isolated mitochondria or specific inhibitors. Experimental approaches to study the import of proteins into mitochondria are also discussed.

Animals↗
PubMed1985

A synthetic signal peptide blocks import of precursor proteins destined for the mitochondrial inner membrane or matrix.

A peptide corresponding to amino acids 1-27 of preornithine carbamyltransferase (pOCT) has been chemically synthesized. When added to energized mitochondria in vitro, 20 microM of the peptide, designated pO(1-27), resulted in a collapse of the electrochemical potential across the mitochondrial inner membrane. This effect on transmembrane potential was not observed, however, when pO(1-27) was added to energized mitochondria under conditions that support in vitro import of precursor proteins (i.e. in the presence of reticulocyte lysate). The latter finding, therefore, made possible an examination of the ability of pO(1-27) to block import of homologous and heterologous proteins into the organelle. At 5-10 microM, pO(1-27) prevented import of pOCT in vitro; inhibition was overcome by increasing the concentration of pOCT. In contrast, pO(16-27), a peptide corresponding to amino acids 16-27 of pOCT and exhibiting a charge:mass ratio similar to pO(1-27) had no such inhibitory effect. pO(1-27) blocked import of other unrelated precursor proteins destined either for the mitochondrial matrix (pre-malate dehydrogenase and a hybrid protein containing the signal sequence of pre-carbamyl phosphate synthetase) or for the mitochondrial inner membrane (pre-thermogenin).

Amino Acid Sequence↗
PubMed1985

Effect of a cold stimulus on the synthesis of uncoupling protein in brown adipose tissue of rats and newborn rabbits.

Rats are known to respond to a cold stimulus by increasing the activity and amount of the uncoupling protein in brown adipose tissue. A 48 h cold stimulus was found to increase the synthesis of uncoupling protein 3.8-fold in 4-5 week old rats whereas no change was observed with newborn rabbits. The lack of response in the latter case may reflect a difference between rabbits and rats or that synthesis is already maximal in newborn rabbits.

Adipose Tissue, Brown↗
PubMed1985

Immunological studies of the uncoupling protein of brown adipose tissue.

The immunological relationship of the uncoupling protein from brown adipose tissue of several mammalian species was examined by using a rabbit antibody preparation against the rat protein. Complete cross-reactivity of the antibody to the protein from hamster, mouse, and rat was found, whereas the protein from rabbit cross-reacted only 25%. Cross-reactivity was also found with the human uncoupling protein, although the human protein was found to be about 1 kdalton smaller than the rat protein. No protein of the size of the uncoupling protein was detected in several tumor cell lines examined.

Adipose Tissue, Brown↗
PubMed1985

Size analysis of uncoupling protein and its precursor from brown adipose tissue of different species.

The apparent size of the monomeric form of uncoupling protein from brown adipose tissue of several mammalian species was compared by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Including earlier results the apparent molecular mass of the protein from rat was about 32 000 daltons and varied about 1500-2000 daltons from the different sources with the size increasing in the order human less than rat less than or equal to mouse less than hamster less than or equal to rabbit. The size of newly synthesized uncoupling protein was also found to vary among species. However in the two cases examined, rat and rabbit, the precursor and its respective mature monomeric protein had the same apparent size as shown by coelectrophoresis.

Adipose Tissue, Brown↗
PubMed1985

Immunohistochemical identification of the uncoupling protein in rat brown adipose tissue.

Brown adipose tissue mitochondria are characterized by the presence of an uncoupling protein that gives them an exceptional capacity for substrate-controlled respiration and thermogenesis. The specific localization of this protein in rat brown adipocytes was demonstrated using an immunohistochemical technique, the peroxidase-antiperoxidase (PAP) method. Light microscopy observations showed that serum antibodies raised against the uncoupling protein selectively reacted with multilocular brown adipocytes. No labeling could be detected in either unilocular adipocytes, capillaries, or muscle fibers (striated and vascular smooth muscle). Staining was more intensive in certain adipocytes than in others, suggesting the presence of cellular heterogeneity. The specificity of the staining technique was demonstrated by showing that treatment of the preparations with antiserum saturated with an excess of uncoupling protein almost entirely inhibited brown adipocyte labeling. The specificity and selectivity of the PAP method allow the clear differentiation of uncoupling protein-containing adipocytes from other cellular types, suggesting that this immunohistochemical technique will represent an extremely useful tool for studying adipocyte function and differentiation.

Adipose Tissue, Brown↗
PubMed1984

Import of rat liver mitochondrial malate dehydrogenase. Binding of the precursor to mitochondria, an intermediate step in import.

We have previously reported that the precursor of rat liver mitochondrial malate dehydrogenase, synthesized in vitro, is about 1,500 to 2,000 Mr larger than the mature enzyme and can be processed to the mature size by isolated mitochondria from Chinese hamster ovary cells (Chien, S.-M. and Freeman, K. B. (1984) J. Biol. Chem. 259, 3337-3342). Furthermore, binding, but not processing, was observed in the presence of an uncoupler. Binding was insensitive to temperature and was completed within 2.5 min at 0 degrees C. The role of binding in the overall process of import of the precursor is now further characterized. The precursor form, bound either in the presence of an uncoupler or at 0 degrees C, was sensitive to trypsin suggesting that binding occurs on the mitochondrial outer membrane. Saturation of binding was observed with a limited amount of mitochondria and an excess of in vitro translated rat liver proteins indicating that there is a finite number of binding sites. Furthermore, when the precursor was prebound to mitochondria at 0 degrees C for 5 min, the precursor was processed to the mature size and the rate of processing was independent of the volume of reaction mixture. In contrast, the rate of processing of unbound precursor was dependent on reaction volume. These results strongly suggest that binding of the precursor of malate dehydrogenase to the mitochondrial outer membrane is an intermediate step in its import.

Animals↗
PubMed1984

Modulation of membrane fusion by ionotropic and thermotropic phase transitions.

We have studied the relationship of ionotropic and thermotropic phase transitions to divalent cation induced fusion of large unilamellar phospholipid vesicles. Fusion was monitored by the Tb/dipicolinic acid fluorescence assay for the intermixing of internal aqueous contents of vesicles. The phase behavior of the membranes was followed by differential scanning calorimetry. (1) Sr2+ and Ba2+ shifted the phase transition temperature (Tc) of bovine brain phosphatidylserine vesicles from 6 to 27 and 31.5 degrees C, respectively. These cations induced vesicle fusion at temperatures above or below the Tc of that cation/phospholipid complex, indicating that an isothermal phase change from the liquid-crystalline to the gel phase is not a requirement for membrane fusion. (2) The temperature dependence of the initial rate of fusion of phosphatidylserine/dipalmitoylphosphatidylcholine (1:1) vesicles in the presence of Ca2+ exhibited a pronounced maximum at 17 degrees C, at the lower part of the broad phase transition endotherm whose Tc was about 25 degrees C; fusion was inhibited completely at 30 degrees C when the membrane was in the liquid-crystalline state. These observations suggest that molecular clusters rich in phosphatidylserine, formed when the membrane is in the phase transition region, allow the vesicles to fuse. (3) The fusion of phosphatidylserine/dimyristoylphosphatidylethanolamine (1:1) vesicles, whose Tc was also around 25 degrees C, had a different temperature dependence in that the initial rate increased sharply above the Tc, with a local maximum within the transition region. Phase separation of dimyristoylphosphatidylethanolamine was induced by Ca2+ but not by Mg2+, although both ions induced fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Import of rat liver mitochondrial malate dehydrogenase. Synthesis, transport, and processing in vitro of its precursor.

Rat liver total RNA was translated in a reticulocyte lysate, and the precursor of rat liver mitochondrial malate dehydrogenase was identified by a monospecific antibody against the denatured mature enzyme. The precursor is about Mr = 1500 to 2000 larger than the monomeric form of the mature protein. The major spots of the two-dimensional peptide map of the two proteins were identical. The precursor was synthesized on free polysomes, but not membrane-bound polysomes. Upon fractionation by molecular sieve chromatography on Sephadex G-100, the size of the precursor was slightly larger than the dimeric form of the mature protein. Incubation of the precursor with isolated mitochondria from Chinese hamster ovary cells resulted in uptake and processing of the precursor to the mature size. The processed form was resistant to trypsin indicating that it was translocated into mitochondria. Processing was complete in 10 to 30 min at 30 degrees C. Rapid binding of the precursor to mitochondria was also observed at 0 or 30 degrees C. Processing but not binding was inhibited by an uncoupler.

Animals↗
PubMed1984

Biosynthesis of the 32-kdalton uncoupling protein in brown adipose tissue of developing rabbits.

The biosynthesis of the 32-kdalton uncoupling protein of brown adipose tissue was examined with polysomes isolated from brown adipose tissue of developing rabbits. Polysomes were translated in a reticulocyte protein-synthesizing system and newly synthesized uncoupling protein was isolated with monospecific antibodies. The newly synthesized protein had the same molecular mass as the mature protein. Its synthesis is initiated and increases about five- to seven-fold before birth and decreases progressively after birth.

Adipose Tissue, Brown↗
PubMed1983

Characterization of a Chinese hamster ovary cell line resistant to uncouplers.

The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK-) cells resistant to 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), a potent uncoupler of oxidative phosphorylation. About 9-times and 4-times more S-13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK- cells. UH5 cells were cross-resistant to the uncouplers SF-6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile), carbonylcyanide p-trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK- cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler-sensitive cell line were sensitive to S-13 indicating that resistance is probably nuclear-determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+-ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK- cells. The oligomycin-sensitive H+-ATPase activity of UH5 and CHO TK- cell mitochondria was equally stimulated by the uncoupler S-13. Uncoupler-resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered.

Adenosine Triphosphatases↗
PubMed1983

Synthesis in vitro of rat brown adipose tissue 32 000 Mr protein.

The synthesis of the mitochondrial inner membrane 32 000 Mr 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 Mr 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.

Adipose Tissue, Brown↗
PubMed1981

Cybrid formation with recipient cell lines containing dominant phenotypes.

A clone of Chinese hamster ovary (CHO) cells, BT3, resistant to Tevenel, the sulfamoyl analog of chloramphenicol has been isolated. Resistance was found to be at the mitochondrial level and was shown to be cytoplasmically inherited. This marker was then used to develop a method by which a cell line possessing a dominant nuclear mutation (resistance to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, DRB) could be used as the recipient in cybrid formation. The unique feature in this procedure was the removal of nucleated cells from the cytoplasts by passage through unipore filters. The dominant character of the DRB- and Tevenel-resistant phenotypes permitted the selection of cybrids immediately after fusion. This initially increased the frequency of cybrid clones 16-fold as compared to a recipient cell line possessing a recessive marker. The possibility of extending the method to recipient cells lacking a selectable drug-resistance marker is discussed.

Animals↗