Ribonucleic acids from barley leaves

1. The total RNA and the RNA present in 27000g pellet (probably composed of chloroplasts, nuclei and mitochondria) and in 27000g supernatant (probably composed of microsomes and soluble proteins) fractions (separated by centrifugation at 27000g of a leaf homogenate prepared in 0·5m-sucrose–0·02m-tris–HCl, pH7·6) of barley leaves were extracted by phenol–sodium lauryl sulphate and their elution profiles on Sephadex G-200 and on ECTEOLA-cellulose anion-exchanger were examined and their nucleotide compositions and the melting curves were determined. 2. The pellet and the supernatant fractions contained respectively about 55% and 20% of the total RNA, whereas 25% of the total RNA was lost during homogenization of the leaf tissue with sucrose–buffer. 3. The total RNA or the RNA from pellet or supernatant fractions, which by its behaviour on Sephadex G-200 columns was found to be predominantly of high molecular weight (i.e. of ribosomal origin), produced about 13 peaks on ECTEOLA-cellulose columns. The RNA species in the pellet and supernatant fractions probably resembled each other in molecular size or secondary structure or both. However, they were present in relatively different amounts in these fractions. 4. The T_m (i.e. the temperature at which 50% of the maximal increase in extinction had occurred) of total RNA and of RNA from pellet fraction was 64·5° whereas T_m of RNA from the supernatant fraction was 73°. The total RNA and the RNA from pellet fraction also resembled each other in nucleotide composition, and the RNA from the supernatant fraction in accordance with its high T_m had a high GMP+CMP content.     

Purification and Characterization of a Keratinase from a Feather-Degrading Bacillus licheniformis Strain

A keratinase was isolated from the culture medium of feather-degrading Bacillus licheniformis PWD-1 by use of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchange and Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purified keratinase is monomeric and has a molecular mass of 33 kDa. The optimum pH and the pI were determined to be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was 50°C. The enzyme is stable when stored at −20°C. The purified keratinase hydrolyzes a broad range of substrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase is a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal.     

Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5′-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg²⁺ and Ca²⁺, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca²⁺ was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50°C on DNA and at 60°C on RNA and attacked RNA and DNA exonucleolytically and successively, producing 5′-mononucleotides.     

A study of the effects of reaction with formaldehyde on some optical and physical properties of reticulocyte ribosomes

1. The optical rotatory dispersion and ultraviolet-absorption spectrum of ribosomal RNA in situ appear to be unchanged when the ribosome is dissociated into its RNA and protein moieties. 2. Reaction with 0·05% formaldehyde at 20° for 2hr. `fixes' ribosomes so that they remain intact in 1% sodium dodecyl sulphate. 3. The RNA moiety of the ribosome undergoes a conformational change when ribosomes in 8% formaldehyde are heated at 70° for 10min. and cooled to 20°. After this treatment no double-helical character can be detected, but neither the sedimentation coefficient nor the morphology of the ribosome determined by electron microscopy is altered. 4. It is concluded that the RNA moiety of reticulocyte ribosomes is freely accessible to formaldehyde.     

Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-β-N-acetylglucosaminidase

On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5±0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6±0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000±5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune, 1970; Wadström, 1970). These results indicate that this extracellular enzyme which is produced by several strains of S. aureus is not a `lysozyme' (endo-β-N-acetylmuramidase) as previously suggested, but an endo-β-N-acetylglucosaminidase.     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.     

Enzymes and cancer: Preparation and some properties of guanase from rabbit liver

1. Guanase has been purified 200-fold in 20% yield from the supernatant fraction of rabbit-liver homogenates, by using ammonium sulphate fractionation, calcium phosphate-gel adsorption and chromatography on DEAE-cellulose and Sephadex G-200. 2. K_m with guanine as substrate at the optimum pH of 7·7 was found to be 1·05×10⁻⁵m. Q₁₀ was 1·4 between 23° and 48°. 3. Substrate activity and pH optima of compounds related to guanine have been studied. 8-Azaguanine, 1-methylguanine, thioguanine and 1-methylthioguanine are all substrates.     

Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase

1. The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. 2. An alkaline (pH10·5) extraction of cytokinase from rabbit kidney lysosome–microsome fraction, followed by chromatography on DEAE-cellulose at pH7·6 with stepwise or linear increase in concentration of phosphate buffer, gave an 86-fold purification of the enzyme. The purified material was non-proteolytic against casein and heated fibrin and was freeze-dried without significant loss of activity or solubility. 3. Cytokinase is a protein with E^0·1%_1cm.=0·87 at 280mμ, and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S_20,w 2·9–3·1s and molecular weight 50000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH8 and pH9, and is maximally active and stable at pH8·5. It is inactivated by heat at 78°. 4. Cytokinase and human urokinase have the same K_m value and are inhibited in a partially competitive manner by -aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. 5. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.     

IUB commission of editors of biochemical journals the citation of bibliographic references in biochemical journals recommendations (1971)

A pentose-rich acidic glycoprotein was isolated from protease digested bovine vitreous humor by fractionation on an AG1-X2 column using NaCl solution gradient.The material eluted at 0.35 M NaCl (glycoprotein) was electrophoretically heterogeneous at pH 8.6 after partial purification on Sephadex G-25. Gel filtration on G-100 resolved the glycoprotein into two fractions. These fractions differ in molecular weight; mol. wt approx. 95 000 material consisted of two components on electrophoresis and mol. wt approx. 28 000 material showed only a single component on electrophoresis. The lower molecular weight component was re-chromatographed on Sephadex G-100 yielding a single orcinol positive component which gave a homogeneous band on gel electrophoresis.Quantitative analysis of this material gave 30% protein, 7.0% pentose, 18.7% glucosamine, 9.2% galactosamine, 10.9% hexuronic acid and 16.1% hexose.Treatment with 0.5 M NaOH at 20°C for 24 h resulted in a 50% decrease in the threonine content suggesting the possible involvement of this amino acid in the protein-carbohydrate linkage group.Paper chromatography of the fraction hydrolysate demonstrated the presence of glucurone, xylose, arabinose, glucose and galactose.     

Isolation, Purification, and Some Properties of Penicillium chrysogenum Tannase

Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The K_m value was found to be 0.48 × 10⁻⁴ M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels.     

Alkaline phosphatase from pig kidney. Method of purification and molecular properties

Alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes was solubilized from membrane precipitates by butan-1-ol at a critical pH of 7.0. The 12000-fold purification procedure included (NH₄)₂SO₄ precipitation, DEAE-and TEAE-cellulose chromatography, Sephadex G-200 gel filtration and neuraminidase digestion followed by DEAE-cellulose chromatography. The purified protein contained 20% (w/w) carbohydrate and had mol.wt. 150000–156000 as estimated by Sephadex filtration and ultracentrifuge analysis. It was a tetrameric glycoprotein consisting of identical subunits, and it had a molecular activity at 25°C of 2600s⁻¹ per tetramer. Its concentration in kidney was estimated to be 8.5–8.8mg/kg.     

Partial Purification and Characterization of a Thermostable Actinomycete β-Amylase

A thermostable amylase, possibly a β-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60°C and pH 7 and by retention of 70% activity at 70°C (30 min). It was stimulated by Mn²⁺ and Fe²⁺ but strongly inhibited by Hg²⁺. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.     

A study of the glucosamine-containing constituents of the seeds of kidney beans (Phaseolus vulgaris)

1. The constituents of the seeds of kidney beans containing glucosamine that could be released by acid hydrolysis (0·5 n-hydrochloric acid at 100°) were extracted into the phenol-rich phase on partitioning between phenol and water at 70°. 2. These materials were also brought into solution on extracting the seeds with water (incomplete), or preferably with a slightly alkaline buffer in the absence of phenol. When these solutions were heated at 100° or treated with trichloroacetic acid, all the materials containing acid labile glucosamine were carried down with the precipitate. Treatment with pepsin rendered the materials containing glucosamine soluble again. 3. All proteins in these extracts of kidney bean were found to be associated with various amounts of neutral and amino sugars by high-voltage electrophoresis and chromatography on Sephadex G-200 columns. 4. The isolation and crystallization of the main sugar components, d-glucosamine and d-mannose, are described and their significance in plant glycoproteins is discussed.     

Indirect Measurement of the Lag Time Distribution of Single Cells of Listeria innocua in Food

The distribution of log counts at a given time during the exponential growth phase of Listeria innocua measured in food samples inoculated with one cell each was applied to estimate the distribution of the single-cell lag times. Three replicate experiments in broth showed that the distribution of the log counts is a linear mapping of the distribution of the detection times measured by optical density. The detection time distribution reflects the lag time distribution but is shifted in time. The log count distribution was applied to estimate the distributions of the lag times in a liquid dairy product and in liver paté after different heat treatments. Two batches of ca. 100 samples of the dairy product were inoculated and heated at 55°C for 45 min or at 62°C for 2 min, and an unheated batch was incubated at 4°C. The final concentration of surviving bacteria was ca. 1 cell per sample. The unheated cells showed the shortest lag times with the smallest variance. The mean and the variance of the lag times of the surviving cells at 62°C were greater than those of the cells treated at 55°C. Three batches of paté samples were heated at 55°C for 25 min, 62°C for 81 s, or 65°C for 20 s. A control batch was inoculated but not heated. All paté samples were incubated at 15°C. The distribution of the lag times of the cells heated at 55°C was not significantly different from that of the unheated cells. However, at the higher temperatures, 62°C and 65°C, the lag duration was longer and its variance greater.     

Purification and properties of diaminopimelate decarboxylase from Escherichia coli

1. Diaminopimelate decarboxylase from a soluble extract of Escherichia coli A.T.C.C. 9637 was purified 200-fold by precipitation of nucleic acids, fractionation with acetone and then with ammonium sulphate, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose or DEAE-Sephadex. 2. The purified enzyme showed only one component in the ultracentrifuge, with a sedimentation coefficient of 5·4s. One major peak and three much smaller peaks were observed on electrophoresis of the enzyme at pH8·9. 3. The mol.wt. of the enzyme was approx. 200000. The catalytic constant was 2000mol. of meso-diaminopimelic acid decomposed/min./mol. of enzyme, at 37°. The relative rates of decarboxylation at 25°, 37° and 45° were 0·17:1·0:1·6. At 37° the Michaelis constant was 1·7mm and the optimum pH was 6·7–6·8. 4. There was an excess of acidic amino acids over basic amino acids in the enzyme, which was bound only on basic cellulose derivatives at pH6·8. 5. The enzyme had an absolute requirement for pyridoxal phosphate as a cofactor; no other derivative of pyridoxine had activity. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was also needed as an activator. 6. In the presence of 2,3-dimercaptopropan-1-ol (1mm), heavy-metal ions (Cu²⁺, Hg²⁺) did not inhibit the enzyme, but there was inhibition by several amino acids with analogous structures to diaminopimelate, generally at high concentrations relative to the substrate. Penicillamine was inhibitory at relatively low concentrations; its action was prevented by pyridoxal phosphate.     

The transporting proteins of cholecalciferol and 25-hydroxycholecalciferol in serum of chicks and other species. Partial purification and characterization of the chick proteins

Chick serum contains two cholecalciferol-binding proteins, one of which binds mainly cholecalciferol (cholecalciferol-binding protein) and the other binds 25-hydroxycholecalciferol (25-hydroxycholecalciferol-binding protein). By means of Cohn fractionation, (NH₄)₂SO₄ precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephadex and an additional gel-filtration step on Sephadex G-100, these two binding proteins were purified. Both proteins possess β-globulin mobility on analytical polyacrylamide-disc-gel electrophoresis, a sedimentation coefficient of 3.5S and approximate molecular weights of 60000 for the cholecalciferol-binding protein and 54000 for the 25-hydroxycholecalciferol-binding protein. Sera obtained from rat, pig, human and monkey were shown to contain a single binding protein that is responsible for the transport of both cholecalciferol and 25-hydroxycholecalciferol. In the toad the lipoproteins are used for the transport of these two steroids.     

Some Properties of Phytochrome Isolated From Dark-grown Oat Seedlings (Avena sativa L.)

Phytochrome was partially purified from etiolated seedlings of Avena sativa L. Several properties of the red-absorbing (P_R) and far-red absorbing (P_FR) forms of the pigment were compared. The 2 forms could not be shown to differ with respect to their sedimentation velocity in sucrose density gradients, elution volume from Sephadex G-200 columns, binding properties on calcium phosphate, or electrophoretic mobility. P_FR, however, was more labile than P_R during precipitation with 50% ammonium sulfate. Sephadex G-200 elution diagrams obtained with fresh phytochrome preparations revealed 2 components of different molecular weights, 1 roughly 180,000, and 1 roughly 80,000. Native phytochrome had an absorption spectrum in vivo showing an absorption maximum for P_R of 667 nm. Both the large and small forms of phytochrome mentioned above can be maintained with an absorption maximum for P_R of 667 nm. However, allowing them to remain for several hours as P_FR, even at 4°, shifted this peak to 660 nm. The protein conformational change during phytochrome transformation may be quite small, though the various comparative techniques used do not strictly rule out a fairly large one. The need for maintaining the pigment as P_R during all steps of purification, but particularly during ammonium sulfate precipitation is underscored.     

Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M_r 30,000). The enzyme had a K_m for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37°C for 1 hour, (NH₄)₂SO₄ fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M_r 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37°C for 1 hour.     

Purification and physical properties of sweet-almond α-galactosidase

1. α-Galactosidase from sweet almonds was purified about 2000-fold through eight steps. 2. The enzyme preparation was free from other related enzymes known to occur in sweet almonds, and behaved as a homogeneous protein on filtration through Sephadex G-75. 3. A molecular weight of about 33000 was determined from the gel-filtration data. 4. The ultraviolet-absorption spectrum and thermal inactivation of the enzyme are described. 5. The purified enzyme hydrolysed p-nitrophenyl α-d-galactoside at a much faster rate than melibiose. 6. The pH optimum was at 5·5–5·7. 7. Besides hydrolysis, it also catalysed transfer of galactosyl residues, chain elongation of melibiose and the synthesis of oligosaccharides from galactose.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.