Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increase in histone acetylation and transitions in histone variants during Friend cell differentiation

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.     

Characterization of lamin proteins in BHK cells

Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.     

Degradation of soluble collagen by ozone or hydroxyl radicals

Collagen exposed to ozone or hydroxyl radicals was degraded in a time- and dose-dependent manner. This degradation was inhibited by free radical scavengers. Furthermore, lower levels of these oxidants did not degrade the molecule, but caused it to become susceptible to proteolytic degradation. We suggest an alternative mechanism by which oxygen-derived free radicals participate in the destruction of extracellular matrix observed during acute lung injury by oxidant gas, in addition to the commonly accepted proteinase-antiproteinase theory of lung injury.     

Fasciola hepatica: tegumental alterations as a consequence of lectin binding

Adult flukes, Fasciola hepatica, incubated in Hedon - Fleig saline containing concanavalin A (Con A) for 10 and 45 min, respectively, exhibited severe alterations to tegumental morphology involving increased secretory activity, blebbing of the apical plasma membrane, increased total surface area, and swelling of the basal infolds . The effects of Con A were prevented by the addition of alpha-methyl-D-mannoside to the incubating medium. Similar, but less pronounced, effects were caused by wheat germ agglutinin (WGA) binding. Con A and WGA binding indicate the presence of mannose, glucosamine, or glucose moieties and of N-acetylglucosamine. The effects of lectin binding were similar to the early effects of antibody attachment, and it was considered that accelerated membrane turnover was occurring in both cases. Swelling of the basal infolds was thought to be a result of increased apical surface membrane and/or increased permeability due to lectin binding.     

Relationships between the effects of redox potential, alpha-thenoyltrifluoroacetone and malonate on O(2) and H2O2 generation by submitochondrial particles in the presence of succinate and antimycin

A successful approach has been developed for the sequencing of apolipoprotein B based upon the procedure of Cleveland et al. [(1977) J. Biol. Chem. 252, 1102-1106] involving limited proteolysis in the presence of sodium dodecyl sulfate. Staphylococcus aureus protease was employed to produce large peptides which were isolated in relatively pure form by preparative gel electrophoresis. Two peptides were partially sequenced using spinning-cup microsequencing techniques. The sequences are: Peptide R2-5, -Ala-Leu-Val-Gly-Ile-Asn- Gly-Glu-Ala-Asn-Leu-Asp-Phe-Leu-Asn-Ile-Pro-Leu-Arg-Ile-Pro-Pro- Met-Arg-(Arg)-; Peptide R3-1, -Leu-Val-Ala-Lys-Pro-Ser-Val-Ser-Val-Glu- Phe-Val-Thr-Asn-Met-Gly-Ile-Ile-Pro-Lys-Phe-Ala-Arg-. Several stretches of residues suitable for the construction of oligonucleotide probes have been identified.     

Fructose 2,6-bisphosphate, sugar phosphates and adenine nucleotides in the regulation of glucose metabolism in the lactating rat mammary gland

Fructose 2,6-bisphosphate is present in the rat mammary gland, rising from a value of 1.4 nmol/g in pregnancy to 4.3 nmol/g tissue at 14 days lactation; the equivalent values calculated/ml intracellular water are 5.2 and 11.6 nmol, respectively. The tissue content of fructose 6-phosphate, fructose 1,6-bisphosphate, ATP and phosphoenolpyruvate remain relatively constant in the transition from pregnancy to the height of lactation. The changes in AMP, cyclic AMP, and citrate content of the mammary gland during lactation are such as to promote an increase in fructose 2,6-bisphosphate formation and flux through phosphofructokinase.     

The glycoprotein 71 of ecotropic Friend murine leukemia virus. Structure of the oligosaccharides linked to asparagine-12

The glycoprotein from Friend murine leukemia virus was digested with protease from Staphylococcus aureus V8. A glycopeptide comprising the N-terminal glycosylation site (Asn-12) was isolated from the mixture of fragments and analyzed by amino acid sequencing and methylation-capillary gas chromatography-mass spectrometry before and after treatment with sialidase from Vibrio cholerae. Asn-12 was thus found to be substituted by a family of partially sialylated, fucosylated, and intersected glycoprotein N-glycans of the hybrid type.     

Mutant hemoglobin stability depends upon location and nature of single point mutation

The temperature dependence of the rates of heme release from the beta subunits of methemoglobin A and 5 beta mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the alpha 1/beta 2 interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.