Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

Proton-translocating adenosinetriphosphatase in rough and smooth microsomes from rat liver

Rat liver smooth and rough microsomal membranes exhibit an ATP-dependent H+ transport which can be inhibited by sulfhydryl reagents and dicyclohexylcarbodiimide but is resistant to oligomycin. On the basis of inhibitor sensitivities and substrate specificities, this H+ pump was found to be different from that of mitochondria, lysosomes, gastric H+-K+-ATPase, and yeast plasma membrane H+-ATPase but to resemble that of endocytic vesicles and the H+ pump responsible for urinary acidification. The transport process is accelerated by valinomycin in the presence of potassium, suggesting that it is an electrogenic pump. The same fractions were enriched in an ATPase with inhibitor sensitivities similar to those of the transport activity. It is possible that the proton electrochemical gradients generated by this pump may play a role in the translocation of proteins and sugars, two of the major functions of these structures.     

Highly purified ceruloplasmin messenger RNA from rat liver

Summary Highly purified ceruloplasmin mRNA was isolated from rat liver polyribosomes. The molecular weight of ceruloplasmin mRNA is in a range from 1.05 to 1.25 · 10⁶ daltons which is large enough to code for a putative precursor of ceruloplasmin (∼700 amino acids). Ceruloplasmin mRNA contains 3′-terminal poly(A) the length of which varies from 38 to 165 nucleotides. The 5′-end of ceruloplasmin mRNA is blocked with confronting m⁷G residue which is a component of cap I (m⁷G^5′ppp^5′X^mpAp). The addition of ceruloplasmin mRNA to wheat-germ cell-free system programmed the synthesis of a product that was largely precipitated by anti-ceruloplasmin immunoglobulins. The translation product was homogeneous in polyacrylamide gel-sodium dodecylsulfate electrophoresis. Cell-free translation of ceruloplasmin mRNA was sensitive to inhibition by cap analogue.     

Saponin permeabilization of rough microsomes from rat liver reveals a novel prothrombin pool

Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following ¹⁴C-γ-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.     

Nuclear precursors for ceruloplasmin-coding messenger RNA from rat liver

The distribution of the sequences coding for ceruloplasmin (CP) in rat liver heterogeneous nuclear RNA (hnRNA) was studied using highly specific CP cDNA as a hybridization probe. The content of CP-coding sequences in poly(A)-containing and poly(A)-free subfractions of hnRNA was shown to be respectively 1 and 27 equivalents of CP mRNA molecule per one hepatocyte. The gel electrophoresis of hnRNA under strongly denaturing conditions with the subsequent transfer of RNA to diazobenzyloxymethyl paper and hybridization with [32P]-cDNA probe showed that CP mRNA sequences were of multiple molecular weight distribution. In particular, 9.0, 6.6, 2.4 and 1.6 megadalton fractions of non-polyadenylate hnRNA carried CP-coding sequences while the only hand that hybridized to CP cDNA was detected in polyadenylated hnRNA. This band was of a molecular weight 1.1-1.2 megadaltons corresponding to that of cytoplasmic CP mRNA. The hybridization of high molecular weight hnRNA with full-length CP cDNA followed by the determination of the size of cDNA fragments protected against SI nuclease demonstrated that coding sequences of CP pre-mRNA are interrupted by intervening sequences.     

Vitamin K epoxidase activity of rough and smooth microsomes from rat liver

The distribution of vitamin K epoxidase activity in rough and smooth microsomes has been studied and compared to the prothrombin precursor and vitamin K-dependent carboxylase activity. All three activities were high in rough microsomes as compared to the low levels found in smooth microsomes. The results are in agreement with the suggestion that there might be a linkage between the vitamin K-dependent carboxylation and epoxidation reaction in vivo.     

Isolation and partial purification of ceruloplasmin messenger RNA from rat liver

Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1×10⁶ daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cell-free translation had molecular weights in the range 4.5–5.4×10⁴ daltons as estimated by gel-electrophoresis under denaturating conditions. These values approach the weight of the half-molecule of native ceruloplasmin.     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.     

Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes

Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

Intramembranous arrangement of the glycosylating systems in rough and smooth microsomes from rat liver

The distribution of mannosyl-, glucosaminyl- and glucosyltransferases in rough and smooth microsomes isolated from rat liver homogenate has been investigated. Amphomycin and tunicamycin were used as inhibitors of dolichol-mediated glycosylation, and diazobenzene sulfonate and proteolytic enzymes were used as nonpenetrating surface probes. Under in vitro conditions only 20-30% of the proteins glycosylated are of the secretory type. Nonpenetrating surface probes, which interact with components on the outer surface of rough microsomal vesicles, decrease glycosylation of both secretory and membrane proteins to a great extent. Inhibitor sensitive glycosylation is present in both the outer and inner compartments of the microsomal membranes. In contrast, the surface probes and the inhibitors of dolichol-mediated glycosylation do not significantly affect protein glycosylation in smooth microsomes. When dolichol phosphate sugars were used as substrates, instead of nucleotide sugars, the probes used inhibited protein glycosylation in both subfractions. Glycosylation of externally added Lipidex-bound dolichol monophosphate and of ovalbumin were in agreement with the above results. It appears that both rough and smooth microsomes may possess several types of glycosylating pathways. The most prominent of these in rough microsomes under the conditions used is the dolichol mono- and pyrophosphate-mediated glycosylation of endogenous proteins, where the enzymes involved in the initial steps are distributed at the outer surfaces of the microsomal vesicles. The dominating pathway in smooth microsomes appears to function in completion of the oligosaccharide chain of the protein and this process does not involve lipid intermediates and cannot be influenced by nonpenetrating surface probes.