Post-transcriptional regulation of catalase synthesis in rat liver and hepatoma: binding of inhibiting factor(s) with polyribosomes synthesizing catalase

Further studies were made on the actions of the inhibiting factor(s) (F_inh) and activating factor(s) (F_act) on catalase synthesis by polyribosomes of hepatoma and normal liver. The following results indicated that F_inh binds with polyribosomes synthesizing catalase. (1) The ribosomal wash of hepatoma, released by treating hepatoma polyribosomes with 0.8 M-KCl, contained F_inh, whereas a similar fraction from normal liver polyribosomes did not. (2) F_inh was not recovered in the ribosomal wash of hepatoma polyribosomes after the latter had been incubated with normal liver supernatant, which contains more F_act than F_inh. (3) The ribosomal wash of normal liver polyribosomes which had been incubated with hepatoma supernatant contained F_inh. This seems to be supported by the finding that in the presence of the supernatant of normal liver treated at pH 5, in which F_act has been inactivated, normal liver polyribosomes can synthesize catalase, whereas hepatoma polyribosomes cannot.F_inh from the supernatant fraction and from the ribosomal wash of hepatoma were indistinguishable in their behaviors on gel filtration on Sephadex G100 and column chromatography on DEAE-cellulose.The specific activities of F_inh recovered in the ribosomal wash fractions from hepatoma and normal liver after incubation with hepatoma supernatant were 100-fold that of F_inh in hepatoma supernatant.It is concluded from these results that catalase synthesis is controlled by F_inh and F_act as follows: F_inh depresses catalase synthesis by binding with polyribosomes synthesizing catalase. F_act restores the synthesis by interrupting the binding of F_inh with polyribosomes, or inactivating F_inh bound to polyribosomes.     

Biosynthesis of Streptomycin

Investigation was carried out on demonstration of two substances constructing a precursor system located at a late stage of streptomycin biosynthesis by Streptomyces griseus. One of them is thought to be a natural precursor of Streptomycin(L) and the other is suggested as an enzymatic substance(H) transforming L to streptomycin. Both substances had no antibiotic potency and H was inactivated at low pH. L was obtained from a cell-free supernatant (active supernatant) prepared from suspension of young mycelium of Streptomyces griseus in glucose solution. H was obtained not only from active supernatant but also from cell-free extract of the organism.

Two ways of isolation were established for L. Active supernatant was adsorbed on a CM-cellulose column equilibrated with 0.05 m Tris-maleate buffer (pH 8.0). Elution of this column with the same buffer as was used for equilibration gave L-containing fraction separated from streptomycin which was eluted with the buffer including 1% of sodium chloride. L was adsorbed also on active carbon in aqueous solution at neutral pH and liberated from it at acidic pH with 95% methyl alcohol. The former method was useful to separate L from streptomycin, and the latter one was so to concentrate L.

H was isolated by using a column chromatography on DEAE-cellulose. After adsorbing active supernatant or cell-free extract of organism on a column equilibrated previously with the same buffer as above, H was eluted with the buffer including 1% of sodium chloride. Cell-free extract of S. griseus was a better source of H supply than the active supernatant.     

Existence of Mg2+-dependent, neutral sphingomyelinase in nuclei of rat ascites hepatoma cells

A sphingomyelinase, which specifically hydrolyzes sphingomyelin into ceramide and phosphocholine, was solubilized from nuclear matrix fraction of rat ascites hepatoma, AH7974 cells. The solubilized enzyme was subjected to Mono Q column chromatography in an FPLC system. The sphingomyelinase which was adsorbed on the column and eluted at 0.25-0.5 M NaCl was characterized. The enzyme required 10 mM MgCl2, 0.01% Triton X-100, 1 mM dithiothreitol, and a higher concentration of buffer than 1 M for its maximal activity, and the optimal pH was 6.7-7.2 in 2 M Tris/acetic acid or 7.5 in 2 M potassium acetate/acetic acid. N-Ethylmaleimide completely inhibited the enzyme activity at 0.2 mM. Therefore, this enzyme is classified as a Mg2+-dependent, neutral sphingomyelinase. The sphingomyelinase sedimented at 4.3S through a 10-30% glycerol gradient containing 2 M potassium acetate. This enzyme was highly specific to sphingomyelin and did not hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Various characteristics of the nuclear sphingomyelinase were similar to those of the plasma membrane enzyme except its requirement for a high concentration of buffer and SH-reagent.     

Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells. The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II₂, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons. The location of GP II₂on the cell surface was confirmed by the fact that it could be labeled metabolically with, D-(³H) glucosamine and externally through the nonpenetrating periodate-NaB³H₄ system. GP II₂could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II₂could be an integral protein. Analysis of the carbohydrate composition of GP II₂ revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked. Finally, GP II₂has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.     

Purification and hydrophobic properties of the δ-endotoxin in the parasporal crystal produced by Bacillus thuringiensis var. entomocidus

Parasporal crystals of Bacillus thuringiensis var. entomocidus were separated from spores and other cell debris by the water-chloroform biphase procedure. The solubilization and fractionation were carried out under mild conditions at 4°C. Crystals were solubilized in 0.01 M dithiothreitol and 0.2 M glycine NaOH buffer at pH 10.0. The solution was treated overnight with 0.01 M Tris-HCl buffer, pH 5.5, containing 0.1% Triton N-101 and 0.1% sodium cholate, and then placed on a Sepharose 6B column, equilibrated, and later developed with the same buffer. Under these conditions, four fractions were obtained, one of which had a molecular weight ranging from 60,000 to 70,000, and demonstrated a high insecticidal activity on second instar larvae of Spodoptera litioralis. The LC₅₀ value of this fraction was a half of that of the solubilized crystals. The other three fractions had a lower activity. The active fraction was further fractionated on an octyl-Sepharose 4B resin. Elution of this column with the same buffer separated the proteins into two fractions. The first eluted fraction was highly active, while the second demonstrated a very low activity. The active fraction was further purified by loading on a short column of octyl-Sepharose 4B and eluted with a linear gradient of the same detergents. Under these conditions, the highly active fraction gave a sharp and symmetrical peak that revealed five close bands at the pH range of 6.1–6.5 on isoelectric focusing gel electrophoresis.     

Phosphoenolpyruvate carboxylase in lupin nodules and roots. Identification of the enzymatic forms

Phosphoenolpyruvate carboxylase (PEPC) EC 4.1.1.31 was extracted from nodules and roots of 2-day-old seedlings of lupin (Lupinus luteus L.). Chromatography on DEAE-cellulose of the nodule extract gave two forms of the enzyme: PEPC I and PEPC II eluted at 0.3-0.35 M and 0.41-0.53 M Tris buffer, respectively. A third form PEPC III from lupin roots was eluted from DEAE-cellulose column at the same buffer concentration as PEPC II from nodules. PEPC I and PEPC II eluted at 0.3-0.35 M and 0.41-0.53 M Tris buffer, more active in the 6-week-old nodules binding effectively nitrogen than in the 12-week-old ones.     

On the synthesis and degradation of the multiple forms of catalase in mouse liver

The incorporation of ⁵⁵Fe-labeled ferrous sulfate and ³H-labeled γ-aminolaevulinic acid into the catalase of mouse liver was measured at intervals up to 96 hr after intraperitoneal injection, and the intracellular location of radioactive catalase followed, as well as the distribution of radiolabel between the multiple forms of this enzyme. At 10 min, catalase radioactivity was present in all the cellular fractions studied, but after this time, label began to disappear from the microsomal fraction and from the peroxisomal detergent extract. By comparison, catalase incorporation reached a peak at about 6 hr in the peroxisomal aqueous extract, and rose to a broad peak after about 30 hr in the cytosol fraction. On resolving the multiple forms of catalase in the supernatant fraction by electrophoresis, it was found that label first appeared in the fastest moving heteromorph, and appeared sequentially in the other multiple forms over a period of 96 hr.The sequence of degradation of catalase was also studied by examination of residual catalase activity subsequent to the injection of allyl-isopropyl acetamide, a heme synthesis antagonist which blocks catalase synthesis. Blood catalase levels did not seem to be significantly affected by this treatment, but in the liver, the decay rates of catalase activity were appreciable, and varied significantly between the intracellular pools. The rate of decrease was greatest in the peroxisomal detergent extract, and least in the supernatant fraction.These findings have been discussed in relation to current understanding of the subcellular disposition, multiplicity, and turnover of hepatic catalase.     

Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria) seeds

The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized. The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer. After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer. After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer. After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg. The RIP was designated lagenin. It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs. The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.     

Recombinant DNA formation in a cell-free system from Xenopus laevis eggs

A cell-free system is described which formed very high levels of recombinant DNA structures in 4 hr at 26°C. It consisted of a single fraction of a high speed supernatant prepared from an extract of unfertilized eggs of the frog Xenopus laevis. This fraction eluted at 0.16?0.18 M Tris homogenization buffer from a DEAE-cellulose column. When two partially homologous supercoiled DNA molecules of different contour lengths were incubated simultaneously in this system, high levels of heterologous figure eight DNA structures were formed and observed by electron microscopy. Subsequent cleavage of the newly formed figure eight structures with Bam HI and Eco RI restriction endonucleases gave rise to “α structures” and “χ structures.” The observed figure eight structures presumably represent the recombination intermediate predicted by the Holliday model for genetic recombination.     

Microsomal synthesis of cholesterol from squalene, Lanosterol, and desmosterol. Evidence for the presence of two noncatalytic activator proteins in the 105,000 g supernatant fraction from brain, heart, and kidney

The biosynthesis of C₂₇ sterols (used as a generic term for 3 β-hydroxysterols containing 27 carbon atoms) from squalene and lanosterol, of cholesterol from desmosterol, and of lanosterol from squalene by microsomal fractions from adult rat heart, kidney, and brain was investigated. These conversions required the presence of 105,000g supernatant fraction. Heat treatment of the supernatant fractions resulted in a significant loss of their capacity to stimulate the conversion of squalene to sterols, but the capacity to stimulate conversion of lanosterol to C₂₇ sterols and desmosterol to cholesterol was unaffected. The stimulatory activity (for the conversion of all three substrates) of both the heated and unheated supernatant fractions was lost on treatment with trypsin. Thus the soluble fraction appears to contribute at least two essential protein components for the overall conversion of squalene to cholesterol; one a heat labile protein, which functions in the squalene to lanosterol sequence, and the other a heat-stable protein, which is operative in the pathway between lanosterol and cholesterol. Hepatic supernatant factors required for cholesterol synthesis by liver microsomal enzymes function with heart, kidney, and brain microsomal enzymes in stimulating sterol synthesis from squalene and sterol precursors. Moreover, heart, kidney, and brain supernatant fractions prepared in 100 mm phosphate buffer stimulated cholesterol synthesis from squalene and other sterol precursors by liver microsomes. The supernatant fractions of the extrahepatic tissues prepared in 20 mm phosphate buffer lacked the ability to stimulate the biosynthesis of lanosterol from squalene by liver microsomes but were able to stimulate the conversion of lanosterol to C₂₇ sterols or conversion of desmosterol to cholesterol. These findings indicate that the heat-stable protein factor present in the supernatant fractions from extrahepatic tissues is perhaps identical to that in liver, but that the heat-labile factor in extrahepatic tissues, which catalyzes the cyclization of squalene to lanosterol, differs in some respect from that in liver.     

Fractionation of heparin by affinity chromatography on covalently-bound human alpha-thrombin

Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of $\text{N-α-tosyl-L-glycyl-L-prolyl-L-arginine-}\text{p}\text{-nitroanilide}$ hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.     

Cellular factor stimulating DNA synthesis in nuclei isolated from sea urchin embryos

DNA synthesis in isolated nuclei of morula-stage embryos of sea urchin was studied. Embryonic extracts of cleaving embryos (but not unfertilized eggs) stimulated DNA synthesis in the in vitro system. A stimulatory factor was identified which eluted at 0.52 M KCl during chromatography on DEAE-cellulose column. This factor was inactivated by heat treatment and trypsin digestion, and was resolved into three active peaks by gel filtration (Stokes radii of 6.3, 4.6, and 4.1 nm, respectively).     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

Increased content of calmodulin in morris hepatoma 5123 t.c.(h)

The content of calmodulin in the 100,000 × g supernatant and particulate fractions in Morris hepatoma 5123 t.c.(h), assayed by its ability to activate the Ca²⁺-activatable cAMP phosphodiesterase, was significantly higher (about 44%) than that in normal or host liver. Only one peak of calmodulin activity was detected when 100,000 × g supernates (350 mg protein) from sonicated homogenates of normal liver and hepatoma were fractionated on a DEAE cellulose column, eluting at a sodium acetate concentration of 0.65 M. The total calmodulin activity eluted from the hepatoma supernatant was 240% higher than that from nornal liver. The increased content of calmodulin in the hepatoma may contribute to the state of abnormal cell proliferation in this tissue.     

Behavior of rat liver catalase during electrophoresis in a pH gradient.

Investigations were conducted on the distribution of rat liver catalase subsequent to electrofocusing in a pH gradient. Differences were observed depending on the enzyme being extracted from the total mitochondrial fraction, from the supernatant of the homogenate or from purified peroxisomes. Catalase solubilized from the total mitochondrial fraction exhibits an apparent isoelectric point lower than that of catalase derived from the supernatant. Catalase released from purified peroxisomes shows a behavior similar to that of the supernatant catalase. It has been concluded that, in a total mitochondrial fraction, a factor is present that alters the electric charge of the catalase molecule during or after the extraction of the enzyme. This factor is probably associated with lysosomes existing together with peroxisomes and mitochondria in a total mitochondrial fraction. As a matter of fact, the addition of an extract of purified lysosomes to purified peroxisomes or to supernatant will cause a shift towards a more acid pH of catalase distribution subsequent to electrofocalization.     

Nuclear location of ribonuclease H and increased level of magnesium-dependent ribonuclease H in rat liver on thioacetamide treatment

Rat liver nuclei were isolated in aqueous solutions of low ionic strength or anhydrous glycerol. The presence of ribonuclease H (RNase H) [EC 3.1.4.34] activity in the cytoplasm is due to extraction of the nuclear enzyme by buffer and inorganic salts. Two forms of RNase H were separated from rat liver nuclei by affinity chromatography using a DNA-cellulose column. When the RNase H in the wash solution of nuclei with 0.3 M sucrose and in nuclear solution extracted with 0.15 M NaCl were fractionated on a single-stranded DNA-cellulose column, two peaks corresponding to Mn2+- and Mg2+-dependent RNases H were eluted at 0.1 M and 0.2 M NaCl, respectively, and a peak having both RNase H activities was recovered in the wash-through fraction from the column. Among the enzymes from these two fractions in the nuclei, the activity of the Mg2+-dependent RNase H which binds to DNA-cellulose increased several-fold within 24 h of a single injection of thioacetamide. The activities of Mg2+-dependent RNase H extracted with higher-salt solution from the nuclei and recovered in the flow-through fraction from the DNA-cellulose column and the Mn2+-dependent RNase H activities were relatively unaffected by an injection of thioacetamide.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.