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)     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Kinetics of the cooperativity of the Ca2+-transporting adenosine triphosphatase of sarcoplasmic reticulum and the mechanism of the ATP interaction.

The extent of the negative cooperativity with MgATP of the Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum has been studied with various membrane preparations and under various conditions. Preparations studied were fragmented sarcoplasmic reticulum vesicles, deoxycholate-solubilized and fractionated ATPase, triton extracted reticulum, vesicles reconstituted from either detergent, and limited trypsin digests of the reticulum. Conditions studied were suboptimal, optimal, and inhibitory Ca²⁺ concentrations; temperatures from 13 to 46 °C; 1 or 5 mm MgCl₂; 0.1 m KCl, 0.1 m NaCl, or no added salt; and Triton or deoxycholate present in the assay. With preparations in which vesicles could accumulate Ca²⁺ ion, the ionophore A23187 was added to prevent inhibition by internal Ca²⁺ ions. Under all circumstances, the negative cooperativity of MgATP was present (Hill coefficient of 0.2 to 0.8), indicating the persistence of the properties of the enzyme molecule and its lipid environment giving rise to kinetic negative cooperativity. Attempts to measure the number of ATP sites by protection against N-ethylmaleimide inactivation and by binding of an analog suggested, but did not prove, that there was only one specific, active ATP binding site below 0.5 mm. These results are interpreted to be consistent with either of two mechanisms for ATP cooperativity of the Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum: (a) a single, high affinity ATP active site and a second, lower affinity “allosteric” activator site; or (b) a single ATP site which demonstrates two affinities through some kinetic mechanism such as a substrate-induced, slow transition.     

Protein composition of human submandibular secretions

The protein composition of human submandibular saliva obtained from a single donor has been investigated, and 21 proteins have been resolved. On Sephadex G-100, submandibular secretions (unstimulated) separated into four fractions, I, II, III, and IV. Each fraction was analyzed further by isoelectric focusing and disc gel electrophoresis. The major components detected in each fraction along with their isoelectric point (pI) are as follows: I, blood group specific substance (2.3), immunoglobulin A (5.0–6.0), and immunoglobulin G (4.5–6.5); II, albumin (4.9), two glycoproteins (5.0), and acid phosphatase (5.2); III, three phosphoproteins (4.3–4.4), isoamylase 1A (5.9), isoamylase 1B (6.4), unidentified protein (7.1), lysozyme (>10), and a basic protein (>10); and IV, isoamylase 2A (5.9) and isoamylase 2B (6.4). Isoamylase 1A and IB are glycoproteins. Stimulated submandibular secretions were also resolved into four protein fractions by gel filtration. Fraction III, compared with unstimulated secretions, showed the greatest percent increase in protein. Analysis of this fraction by disc gel electrophoresis demonstrated the presence of four protein bands which were not detected in the unstimulated secretion. One of these proteins is tentatively identified as a phosphoprotein and two as basic proteins (pI > 10). The protein composition of submandibular, parotid, and sublingual secretions is compared.     

Δ′-Pyrroline: An intermediary metabolite in the conversion of putrescine to 2-pyrrolidone

Δ′-Pyrroline, an oxidative product of putrescine metabolism, was chemically synthesized and incubated with a rat liver homogenate. The incubation mixture was fractionated on an amino acid analyzer before and after acid hydrolysis. The hydrolyzed sample, in contrast to the unhydrolyzed sample, contained a ninhydrin positive compound that cochromatographed with γ-aminobutyric acid, the product of 2-pyrrolidone acid hydrolysis. Authentic 2-pyrrolidone had the same retention time as the Δ′-pyrroline metabolite when analyzed by high-pressure liquid chromatography. It is concluded that Δ′-pyrroline is an intermediary metabolite in the pathway from putrescine to 5-hydroxy-2-pyrrolidone.     

Effect of tunicamycin on the glycosylation of rhodopsin

The incorporation of [³H]glucosamine, [³H]mannose, and [³⁵S]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this well-characterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [³⁵S]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.     

Galactosyltransferase activities in pancreas, liver and gut of the developing rat

Two galactosyltransferase activities (1 and 2) were measured in the pancreas, liver and gut of the developing rat embryo. 1. N-Acetylglucosamine:Galactosyltransferase. UDP [¹⁴C]galactose + N-acetylglucosamine → [¹⁴C]galactosyl-β-(1 → 4)-N-acetylglucosamine + UDP. 2. N-Acetylgalactosamine-protein:Galactosyltransferase. UDP [¹⁴C]galactose + N-acetylgalactosamine-protein → [¹⁴C]galactosyl-β-(1 → 3)-N-acetylgalactosamine-protein + UDP. Galactosyltransferases 1 and 2 increased in the pancreas, about 10- and 40-fold in specific activity, respectively, from 11 to 12 days in utero to birth. During this period the activities of both transferases in the liver were somewhat variable, but showed no definite trend. A drop in the level of galactosyltransferase 1 in the pancreas occurred at birth or shortly thereafter. The “Golgimarker” enzyme for liver, galactosyltransferase 1, may be absent or present at low levels in adult rat pancreas.Zymogen granule membrane preparations apparently are devoid of these galactosyltransferase activities. Bromodeoxyuridine, which inhibits the development of the synthetic capability of the specific exocrine proteins, had essentially no effect on the normal accretion of the galactosyltransferase activities in organ cultures of pancreatic rudiments from 13-day rat embryos.