Demonstration of a precursor-product relationship between soluble and cross-linked elastin, and the biosynthesis of the desmosines in vitro.

Direct evidence showing that a soluble form of elastin is the precursor of cross-linked elastin was obtained from pulse-chase experiments using chick embryo aortas and by demonstrating the conversion of soluble elastin into cross-linked elastin in a cell-free system. Acetic acid extracts of embryonic chick aorta pulse-labeled with [14C]lysine contain two radioactive proteins of molecular weights 74,000 and 138,000 which have been identified previously as soluble elastin and the pro-alpha chain of collagen, respectively. In pulse-chase experiments, the radioactivity incorporated in the soluble elastin during the pulse with [14C]lysine disappeared during a 24-hour chase with [12C]lysine and 89% of that which disappeared was accounted for in the desmosines of alkali-insoluble elastin. The disappearance of the radioactivity from the soluble fraction and its appearance in the desmosines of elastin were inhibited by beta-aminopropionitrile, a specific inhibitor of the cross-linking enzyme lysyl oxidase. In addition in vitro experiments, it was shown that the radioactivity in the desmosines of elastin can arise from that present in an acid-soluble precursor protein. This precursor protein is soluble elastin, as demonstrated by the formation of desmosines when a homogeneous preparation of soluble elastin was incubated with purified lysyl oxidase.     

Assay of protamine messenger RNA from rainbow trout testis.

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.     

Incorporation of L-[1-14C]leucine into protein by liver postmitochondrial supernatant: opposing effects of preincubated nicotinamide-adenine dinucleotide phosphate and 4-dimethylamino-3''-methylazobenzene.

Combination of preincubated drug-metabolizing medium containing NADP+ with a cell-free protein-synthesizing system resulted in marked stimulation of incorporation of L-[1-14C]leucine into protein. Addition of 4-dimethylamino-3'-methylazobenzene, present and previously preincubated in the drug-metabolizing medium, decreased this effect.     

Functional interaction of free polyribosomes with the membrane of the endoplasmic reticulum in a cell-free protein-synthesizing system from plasmacytoma X5563

In cell-free protein synthesis by the murine plasmacytoma X5563, which had become a nonproducing mutant, mixed systems with free polyribosomes and mirosomes incorporated ¹⁴C-amino-acid into protein 3–8 times greater than the sum of the incorporations in the individual system irrespective of S-100 concentrations. This enhancement was inhibited by lecithinase A and was markedly reduced at high KCl concentrations. Smooth endoplasmic membranes had more stimulatory activity than rough endoplasmic membranes. The results indicate that the membrane of the endoplasmic reticulum and free polyribosomes interact in the cell-free protein-synthesizing system, resulting in the enhancement of protein synthesis.     

Inhibition of protein synthesis in Krebs 2 ascites cells and cell-free systems by phenylalanine and its effect on leucine and lysine in the amino acid pool

1. The inhibition of incorporation of ¹⁴C-labelled amino acids into protein of whole cells by phenylalanine has been reproduced in a cell-free system. In both cases only the l-isomer was inhibitory. 2. The effect of phenylalanine on incorporation of [¹⁴C]leucine and [¹⁴C]lysine into protein was different in both whole cells and cell-free systems. 3. In whole cells inhibition of incorporation of leucine at 2·5μg./ml. was very rapid, but when the concentration was increased to 100μg./ml. the inhibition was not apparent for about 1hr. The kinetics of inhibition of lysine was the same at both these concentrations and was similar to that found with leucine at 100μg./ml. 4. Neither a lower specific radioactivity of the two amino acids in the pool nor a decrease in their pool size could be consistently related with inhibition of protein synthesis. 5. In the cell-free system l-phenylalanine inhibited the incorporation of leucine but not of lysine. 6. Charging of transfer RNA by leucine was markedly decreased in the presence of phenylalanine, whereas charging of transfer RNA by lysine was not.     

The transmembrane arrangement of the ADP/ATP carrier as elucidated by the lysine reagent pyridoxal 5-phosphate

The lysine reagent pyridoxal 5-phosphate was applied to the ADP/ATP carrier (AAC) in order to elucidate topological and functional properties of the numerous lysines within the primary structure. To establish appropriate labeling conditions, the influence of pyridoxal-P on transport and inhibitor binding to the AAC was examined. The ADP/ATP transport is sensitive to low concentrations of pyridoxal-P with a Ki = 0.4 mM. Binding of [3H]carboxyatracylate and [3H]bongkrekate is largely inhibited by pyridoxal-P treatment with Ki approximately 1 mM. [3H]Carboxyatractylate is not and [3H]bongkrekate weakly removed by pyridoxal-P, whereas [3H]atractylate is displaced to a large extent. Under optimized conditions of pyridoxal-P concentration, of pH and of time exposure, the AAC was exposed to [3H]pyridoxal-P in mitochondria, in submitochondrial particles and in the detergent-solubilized carrier. The [3H]pyridoxal-P-labeled AAC was isolated from mitochondria and particles. After citraconylation thermolysinolytic peptides were prepared. The pyridoxyl-lysine-containing peptides were purified and the pyridoxal-P incorporation to specific lysines was determined by sequencing. The pyridoxal-P incorporation into the AAC in various states was evaluated with regard to structural and functional aspects. First, by comparing pyridoxal-P incorporation in mitochondria and sonic particles, the segments of the polypeptide chain exposed to the cytosolic and matrix side of the membrane are detected. Second, the additional lysine incorporation into the isolated as compared to the membrane-bound carrier is attributed to the protein collar facing the phospholipid headgroups. Third, the difference between lysine incorporation into the carboxyatractylate-AAC and bongkrekate-AAC complexes reflect either conformational changes or lysines involved in the translocation channel through the protein. Fourth, the additional lysine labeled in the atractylate-carrier complex as compared to the carboxyatractylate-carrier complex is attributed to a cationic site in the binding center. These results are incorporated into a transmembrane folding model of the carrier.     

Biosynthesis of carnitine and 4-N-trimethylaminobutyrate from lysine

The conversion of l-[U-(14)C]lysine into carnitine was demonstrated in normal, choline-deficient and lysine-deficient rats. In other experiments in vivo radioactivity from l-[4,5-(3)H]lysine and dl-[6-(14)C]lysine was incorporated into carnitine; however, radioactivity from dl-[1-(14)C]lysine and dl-[2-(14)C]lysine was not incorporated. Administered l-[Me-(14)C]methionine labelled only the 4-N-methyl groups whereas lysine did not label these groups. Therefore lysine must be incorporated into the main carbon chain of carnitine. The methylation of lysine by a methionine source to form 6-N-trimethyl-lysine is postulated as an intermediate step in the biosynthesis of carnitine. Radioactive 4-N-trimethylaminobutyrate (butyrobetaine) was recovered from the urine of lysine-deficient rats injected with [U-(14)C]lysine. This lysine-derived label was incorporated only into the butyrate carbon chain. The specific radioactivity of the trimethylaminobutyrate was 12 times that of carnitine isolated from the urine or carcasses of the same animals. These data further support the idea that the last step in the formation of carnitine from lysine was the hydroxylation of trimethylaminobutyric acid, and are consistent with the following sequence: lysine+methionine --> 6-N-trimethyl-lysine --> --> 4-N-trimethylaminobutyrate --> carnitine.     

Inhibitors of advanced glycation end product-associated protein cross-linking

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-(14)C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM L-threose, 2.5 microCi [(14)C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0-10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [(14)C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1-0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [(14)C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [(14)C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone.     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism.

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [14C]leucine into protein and had a decreased uptake of [14C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would be consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [14C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Incorporation of arachidonic acid into 1-acyl-2-lyso-sn-glycero-3-phosphocholine of the human neutrophil

In this study, the initial incorporation of arachidonic acid into human neutrophils has been examined. Neutrophils pulse labeled for 5 min with [3H]arachidonic acid rapidly incorporated this fatty acid into 1,2-diacylglycerophosphocholine. However, when neutrophils were pulse labeled with [3H]arachidonic acid for 5 min, washed, and allowed to incubate for an additional 120 min, the relative amount of [3H]arachidonic acid increased in alkylacylglycerophosphocholine molecular species. Similar, when neutrophils were pulse labeled, washed, and allowed to incubate in the presence of 30 microM unlabeled arachidonic acid for 120 min, [3H]arachidonic acid was also remodeled into alkylacylglycerophosphocholine. These results implied that the initial incorporation of [3H]arachidonic acid proceeded via a free fatty acid intermediate into 1,2-diacyl-GPC, while the subsequent remodeling of arachidonate-containing glycerophospholipids did not. This initial incorporation was further investigated in a number of cell-free systems. Disrupted neutrophils incubated with [14C]arachidonoyl-CoA incorporated [14C]arachidonic acid into 1,2-diacyl-GPC containing 16:0, 18:0, and 18:1 at their sn-1 position in a pattern similar to that seen when whole neutrophils were incubated with arachidonic acid for 5 min. A small percentage of [14C]arachidonate from [14C]arachidonoyl-CoA was incorporated into 1-alkyl-2-acyl-GPC. The enzymatic activity responsible was found predominately in the membrane fraction of the broken cell preparation. This selectivity of the CoA-dependent acyltransferase for 1-acyl-linked glycerophosphocholine was further examined by adding [14C]arachidonoyl-CoA and various 1-radyl-2-lyso-GPC to neutrophil membrane preparations. These studies provide evidence that the initial incorporation of arachidonic acid into sn-glycero-3-phosphocholine takes place by an arachidonoyl-CoA: lysophosphatidylcholine acyltransferase(s) which is selective for the 1-acyl-2-lyso-GPC.     

Effect of various aminoacid analogues on chick tendon procollagen synthesis and secretion: Selective inhibition by S-2-aminoethyl cysteine

Matrix-free chick embryo tendon cells were incubated with [¹⁴C]-proline in the presence of various aminoacid analogues and the effects of the analogues on [¹⁴C]-proline incorporation, [¹⁴C]-hydroxyproline synthesis and secretion of labeled molecules were examined. It was found that the structural lysine analogue S-2-aminoethylcysteine was a potent inhibitor of procollagen synthesis and secretion. At a concentration of 1 mM it produced a 75% decrease in [¹⁴C]-hydroxyproline synthesis and a 90% decrease in the secretion of [¹⁴C]-hydroxyproline-containing macromolecules.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.     

Identification of the essential tyrosine residue in the beta subunit of bovine heart mitochondrial F1-ATPase that is modified by 7-chloro-4-nitro[14C]benzofurazan

Inactivation of the bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.5, led to the incorporation of 1.42 g atoms of 14C/mol. Treatment of the inactivated enzyme with dithiothreitol removed 0.99 g atom of 14C/mol of enzyme which was accompanied by reactivation of the ATPase. Therefore, of the 1.42 mol of 7-chloro-4-nitro-[14C]benzofurazan incorporated per mol of bovine heart mitochondrial F1-ATPase, 0.43 mol was present on lysine residues and 0.99 mol was present on tyrosine residues. When the inactivated enzyme was treated with 10 mM sodium dithionite at pH 6.0, 10% of the activity was recovered which was accompanied by a 10% loss in covalently bound 14C. Following dithionite treatment, that part of the 14C which remained covalently bound could not be removed by subsequent treatment of the labeled enzyme with dithiothreitol. It is presumed that dithionite reduces the 4-nitro group of the covalently bound reagent, converting it to 4-amino[14C]benzofurazan derivatives at lysine and tyrosine residues. The moles of 4-amino[14C]benzofurazan incorporated per mol of the isolated subunits were: alpha, 0.18; beta, 0.30; gamma, 0.03; and delta plus epsilon, less than 0.01. Gel filtration of a cyanogen bromide digest of the labeled beta subunit on Sephadex G-75 resolved a major 14C peak which contained 83% of the 14C recovered. The major, radioactive tryptic fragment derived from this peak was purified by gel filtration on Sephadex G-75 followed by reversed phase high performance liquid chromatography. Automatic Edman degradation of this peptide showed that the 14C was released at the position occupied by beta-Tyr-311.     

A photoactivable phospholipid analogue that specifically labels membrane cytoskeletal proteins of intact erythrocytes

A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitro-benzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of [14C]AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. [14C]AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.     

Analysis of translational fidelity of ribosomes with protamine messenger RNA as a template

A novel method was developed to estimate the translational fidelity of mammalian ribosomes in vitro with protamine mRNA of rainbow trout as template. Protamines are mixtures of basic proteins consisting of only seven types of amino acids (Arg, Ile, Val, Ser, Pro, Ala, and Gly), arginine (codon, AGR and CGN) being abundant. Taking advantage of the absence of lysine (codon, AAG) in the proteins, we determined the misincorporation of this amino acid into protamines in a cell-free translation system consisting of mouse liver ribosomes, protamine mRNA, [3H]lysine, [14C]arginine, and seven unlabeled amino acids: Ile, Val, Ser, Pro, Ala, Gly, and Met. After the reaction, translation products were analyzed by either sucrose gradient centrifugation or polyacrylamide gel electrophoresis. In the former method, radioactive protamines are mostly found on monosomes, but not on polysomes, probably because of the basic nature of the proteins. The error frequency was calculated from the molar ratio of [3H]lysine to [14C]arginine incorporated into protamines with an appropriate correction. The frequency was found to be 0.0006-0.002. This method enabled us to determine the frequency of misrecognition of purine bases at the second position of arginine codons in mRNA.     

Lysine metabolism in a barley mutant resistant to S(2-aminoethyl)cysteine

Lysine and S(2-aminoethyl)cysteine (AEC) metabolism were investigated in normal barley (Hordeum vulgare L. cv. Bomi) and a hemozygous recessive AEC-resistant mutant (R906). Feedback regulation of lysine and threonine synthesis from [¹⁴C] acetate was unimpaired in plants of the mutant 3 d after germination. Seeds of Bomi and R906 contained similar total amounts of lysine, threonine, methionine and isoleucine. Concentrations of these amino acids in the soluble fraction of plants grown 6 d without AEC were also similar. The concentration of AEC in R906 plants was less than in the parent variety when both were grown in the presence of 0.25 mM AEC for 6 d. The uptake of [³H]AEC and [³H]lysine by roots of R906 was, respectively, 33% and 32% of that by Bomi roots whereas the uptake of these compounds into the scutellum was the same in both the mutant and its parent. The uptake of [³H]leucine and its incorporation into proteins was also the same in Bomi and R906 plants. These results suggest that a transport system specific for lysine and AEC but not leucine is altered or lost in roots of the mutant R906. AEC is incorporated into protein and this could be the reason for inhibition of growth rather than action as a false-feedback inhibitor of lysine biosynthesis.Abbreviations AEC S(2-aminoethyl)cysteine - LYS lysine - THR threonine     

Effects of lysosomotropic agents on lipogenesis

Chloroquine, quinine, and NH4Cl are lysosomotropic agents which inhibit lysosomal function, apparently by raising the intralysosomal pH. We found that preincubation of cultured human skin fibroblasts with these lysosomotropic agents under serum-free conditions induced about a 10-fold stimulation of lipogenesis. A similar stimulatory effect on the incorporation of 3H2O, [14C]acetate, [14C]pyruvate, [14C]palmitate, and [14C]choline into cellular lipids was observed. The effect was both time and dose dependent, and was reversible. The concentrations of chloroquine, quinine, and NH4Cl resulting in half-maximal stimulation were about 3 microM, 30 microM, and 9 mM, respectively. At these concentrations, stimulation of lipogenesis correlated with impairment of lysosomal function. At a concentration of 10 microM chloroquine, the half-time for maximal stimulation was about 4 h. Most of the [14C]acetate was incorporated into phosphatidylcholine and other cellular lipids; less than 10% was found in cholesterol and cholesterol ester. Nevertheless, incorporation of [14C]acetate into cholesterol showed a chloroquine-induced stimulation parallel to that observed for phospholipids, suggesting that stimulation of both lipogenesis and cholesterogenesis occurred. The stimulatory effect of lysosomotropic agents on lipogenesis appeared to depend on active synthesis of cellular proteins. In the presence of cycloheximide, an inhibitor of protein synthesis; the stimulation was completely abolished.     

Effect of sedimentation through sucrose solutions on the protein-synthesizing ability of rat liver microsomes

1. Centrifugation of the postmitochondrial supernatant of rat liver through 1.0m-sucrose produces particles that have 85-95% less incorporating ability in a cell-free protein-synthesizing system than either ribosomes or microsomes. 2. The incorporation of [(14)C]phenylalanine into protein by particles prepared by sedimentation through 1.0m-sucrose is stimulated about 20-fold by addition of poly U. 3. The content of rapidly labelled RNA of microsomes is decreased during centrifugation through 1.0m-sucrose. 4. It is suggested that degradation of mRNA occurs during the formation of the pellet in the centrifuge tube as a result of a membrane-bound alkaline ribonuclease, after removal of the ribonuclease inhibitor of the soluble fraction.