The stimulation of yeast mitochondrial protein synthesis by low molecular weight cytoplasmic factors: Requirement for intact mitochondria and lack of effect by folate derivatives

• 1.1. Protein synthesis by GTP -supplemented yeast mitochondria is stimulated by a fraction of molecular weight less than 2,000 isolated from yeast high-speed supernatant (S-150).
• 2.2. The low molecular weight fraction works independently of the respiratory chain as the stimulation effect is not cyanide-sensitive.
• 3.3. Stimulation of mitochondrial protein synthesis by cytoplasmic factors is dependent upon the method of mitochondrial isolation.
• 4.4. The low molecular weight stimulatory factor(s) are not reduced folate derivatives which supply formyl groups required for initiation of mitochondrial protein synthesis.

     

Hybridization properties of non-polyadenylated oligo(U)-containing RNA from germinating wheat embryos

• 1.1. Total cytoplasmic RNA of germinating wheat embryos was fractionated by affinity chromatography and separated into non-polyadenylated oligo(U)-containing RNA (A(−)U(+)RNA) and polyadenylated oligo(U)-lacking RNA (A(+)U(−)RNA).
• 2.2. The reassociation kinetics of ³²P-labelled complementary DNA (cDNA) reverse-transcribed from A(−)U(+)RNA shows that this RNA fraction is transcribed from unique DNA sequences of the genome similarly as typical mRNA.
• 3.3. Cross hybridization experiments show no significant sequence homology between the two RNA fractions. Therefore it is concluded that non-polyadenylated oligo(U)-containing RNA of wheat embryo may represent a discrete class of mRNA.

     

Specific biochemical and immunological properties of some water-soluble glycoproteins produced by rat gastric mucosal scrapings in vitro

• 1.1. Two major glycoprotein components were released in vitro by rat gastric mucosal cells after 4 hr incubation with radioisotopes: a high molecular weight fraction with the characteristics of a fucomucin and a low molecular weight fraction, the latter having a higher specific radioactivity than the former.
• 2.2. Pulse chase experiments indicate that several low and high molecular weight glycoproteins are synthesized simultaneously with no precursor-product relationship between them.
• 3.3. Common antigenic determinants, specific to the stomach were found on the 2 fractions, using immunofluorescence, both fractions appeared to be present in the same cells.

     

A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

• 1.1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves.
• 2.2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography.
• 3.3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA.
• 4.4. The three enzymes require either Mg²⁺ or Mn²⁺ for activity.
• 5.5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase.
• 6.6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.

     

Isolation and partial characterization of vitellin from the egg of the giant tiger prawn,penaeus monodon

• 1.1. The results obtained from the present study show that vitellin (yolk protein), a glycolipoprotein derived from the giant tiger prawn, Penaeus monodon, consists of four polypeptides, Ep1, Ep2, Ep3 and Ep4, with molecular weights of 168, 104, 83 and 74 kDa, respectively. This protein was purified using SDS-PAGE and gel-elution.
• 2.2. Antisera against Ep2, Ep3 and Ep4 fractions showed very strong specific binding reactivity when reacted with shrimp ovaries at developing stages.
• 3.3. No crossreaction was observed among Ep2, Ep3 and Ep4 fractions of vitellin. However, anti-Ep2 and anti-Ep3 were demonstrated to be able to react with Ep1 fraction. The homogenates obtained from ripe ovaries and hemolymph of vitllogenesis females showed an immuno-identical pattern to those obtained from egg extracts.
• 4.4. The proteolysis mapping for each fraction showed that Ep1, the high molecular weight fraction, contained Ep2 and Ep3 fragments. These results suggest that Ep1 may be a precursor for Ep2 and Ep3.

     

Characteristics of chromatin release during digestion of nuclei with micrococcal nuclease: Preferential solubilization of nascent rna at low enzyme concentration

• 1.1. Extensive digestion of nuclei with micrococcal nuclease (MNase), commonly used in the analysis of chromatin structure, results in the production of mono- and dinucleosomal chromatin fragments.
• 2.2. Digestion of nuclei from a range of cell types with low enzyme concentrations solubilized high molecular weight polynucleosomal fragments, some ⪢ 22 kb long.
• 3.3. Such digestion conditions also resulted in extensive solubilization of nascent RNA which contributed considerably to the nucleic acid content of the soluble fraction.
• 4.4. We conclude that the contribution of RNA to total nucleic acid content of the soluble fraction should be taken into consideration when nuclei are digested with low concentrations of MNase.

     

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose,hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

• 1.1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile.
• 2.2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue).
• 3.3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose.
• 4.4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus.
• 5.5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Amino acid composition and the protein form of procarboxypeptidase a purified from the pancreas of the sei whale Balaenoptera bolealis

• 1.1. Procarboxypeptidase (W-PCPA) was purified from the pancreas of the sei whale Balaenoptera bolealis.
• 2.2. W-PCPA was obtained as a homogeneous protein in polyacylamide gel disc electrophoresis.
• 3.3. W-PCPA has a molecular weight of 75,000.
• 4.4. Amino acid composition of W-PCPA was compared with that of bovine procarboxypeptidase as A S5 (PCPA-S5).
• 5.5. W-PCPA may be two subunits, and the aggregate form may resemble PCPA-S5.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

In vitro Ca deposition by rat matrix vesicles: Is the membrane association of alkaline phosphatase essential for matrix vesicle-mediated calcium deposition?

• 1.1. Phosphatidylinositol phospholipase C (PI-PLC) treatment of rachitic rat matrix vesicles (MVs) released about 80% of membrane-bound alkaline phosphatase (ALP), AMPase, PPiase into the media.
• 2.2. About 20% hydrolytic activity was not released from MV membranes by PI-PLC treatment.
• 3.3. SDS-polyacrylamide gel electrophoresis and Western blot analysis showed only one immunoreactive protein corresponding to the molecular weight of ALP present in the soluble fraction after PI-PLC treatment.
• 4.4. The specific activity of the released ALP was at least 5-fold higher than the residual activity.
• 5.5. After PI-PLC treatment, MVs also demonstrated an 80% reduction of AMP- or βGP-dependent calcium deposition.
• 6.6. The soluble fraction containing 80% of ALP activity was unable to support calcium deposition. The mixing of the soluble and insoluble fractions after PI-PLC treatment failed to fully restore calcium-depositing activity.

     

Lipoxygenase of Thermoactinomyces vulgaris,purification and characterization of reaction products

• 1.1. A lipoxygenase preparation was obtained from Thermoactinomyces vulgaris and was purified by affinity chromatography on a linoleyl aminoethyl sepharose column.
• 2.2. Two active fractions were obtained.
• 3.3. The fraction obtained by elution with 100 mM borate buffer pH 9.0 was used in the subsequent work.
• 4.4. Th. vulgaris lipoxygenase oxidized linoleic acid into two products: 13-HPOD and 9-HPOD at a ratio of 44 to 56, respectively.
• 5.5. The identification and characterization of the isomers was done by HPLC, I.R. and mass spectrometry.
• 6.6. When arachidonic acid was used as substrate, 15-HPETE and 15-HETE were found to be the main enzymatic products.

     

Lipid agglutinins from coelomic fluid of the earthworm,Lumbricus terrestris

• 1.1. Coelomic fluid of the earthworm, Lumbricus terrestris, contains lipid agglutinins that are primarily glycolipids. They are present in fluid from both immunized (induced) and unimmunized (naturally occurring) worms.
• 2.2. Lumbricus agglutinins partially purified by Folch extraction followed by silicic acid chromatography, were present in all fractions but in highest concentration in acetone and methanol fractions.
• 3.3. Immunodetection revealed agglutinin activity in acetone and methanol fractions, but not in the chloroform fraction.

     

Poly(d(A—T)) dependent RNA polymerase activity after treatment of nuclei with micrococcus nuclease

• 1.1. Rat liver nuclei were incubated with or without 20 units micrococcus nuclease (EC3.1.4.7)/mg nuclear DNA.
• 2.2. The soluble poly(d(A—T)) dependent RNA polymerases were reduced in activity to 15–20% that of the controls after treatment with micrococcus nuclease.
• 3.3. RNA polymerases I plus III activities were completely, RNA polymerase II activity partially reversible on removal of the DNA released into the soluble fraction by treatment of nuclei with micrococcus nuclease.
• 4.4. Inhibitory constants obtained with the solubilized DNA were 17.1 μM and 20.7 μM nucleotide-DNA for RNA polymerases I plus III and RNA polymerase II, respectively. The corresponding inhibitory constants obtained with native salmon DNA were 23.0 μM and 34.4 μM nucleotide-DNA.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

Purification and properties of glycollate oxidase from Lemna minor L.

• 1.1. Glycollate oxidase has been purified to apparent homogeneity from Lemna minor L. grown on medium containing 7mM NO⁻₃.
• 2.2. The enzyme is a highly basic protein with a sub-unit molecular weight of 42,000 and a holoprotein molecular weight of 250,000.
• 3.3. The Lemna enzyme is a flavoprotein with a broad specificity for straight chain α-hydroxy acids, the preferred substrate being glycollate.
• 4.4. It is also competitively inhibited by oxalate and phenyllactate.
• 5.5. A comparison is drawn between the physical properties of glycollate oxidase from a number of higher plants and the degree of sub-unit aggregation in the resulting protomers.

     

Purification and some properties of isocitrate dehydrogenase of a blowfly Aldrichina grahami

• 1.1. NADH-dependent isocitrate dehydrogenase has been purified 110-fold from the crude extract of the flight muscle mitochondria of Aldrichina grahami.
• 2.2. The purification procedure involved Triton X-100 treatment of isolated mitochondria, column chromatography on DEAE-cellulose, Affi-gel blue, and P-cellulose.
• 3.3. The purified enzyme was homogeneous by criteria of the polyacrylamide gel electrophoresis.
• 4.4. The enzyme of the blowfly contains more acidic amino acids and less hydrophobic amino acids than that of pig heart.
• 5.5. The molecular weight was determined to be 330,000 daltons. The subunit construction differs from ghat of mammalian isocitrate dehydrogenase.

     

Lipoxygenase of the thermophilic bacteria thermoactinomyces vulgaris—properties and study on the active site

• 1.1. A lipoxygenase activity was purified from Thermoactinomyces vulgaris and some of its properties were characterized.
• 2.2. The enzyme showed a temperature activity range of 40–55°C with still significant activity over 60°C.
• 3.3. The pH of activity on linoleic acid had a broad range with an optimum at pH 6.0 and a weaker one at pH 11.0.
• 4.4. On arachidonic acid the pattern was narrow bell-shaped with an optimum at pH 6.5.
• 5.5. The purified lipoxygenase from Th. vulgaris showed an apparent K_m of 1 mM and V_max of 0.84 μmol diene/min/mg protein.
• 6.6. It was inhibited by the oxidation products, 9-HPOD and 13-HPOD.
• 7.7. A 160,000 Da molecular weight of the enzyme was determined by molecular filtration. Methionine, tyrosine, tryptophan and cysteine are apparently involved in its activity.

     

Purification and partial characterization of 200 kDa oncofetal antigen from radiation induced murine lymphocytic lymphoma

• 1.1. A 200 kDa glycoprotein (gp200) oncofetal antigen was purified from solubilized membranes of a radiation-induced murine lymphomcytic lymphoma cell line (XR11-5T), grown in syngeneic RFM mice, by successive gel chromatography of the active fraction on lentil lectin agarose, Q- and S-Sepharose and Superose-12 using an FPLC system.
• 2.2. A murine monoclonal antibody 115, produced by the syngeneic immunization of adult male C57BL/6N mice with 12-day mouse fetal cells, was used in a slot blot antibody assay to follow up the active fractions.
• 3.3. The purified glycoprotein has a pi of 5.4.
• 4.4. Treatment of radiolabeled gp200 with neuraminidase caused a slight reduction in size due to the removal of sialic acid groups and a shift in pI to 6.3.
• 5.5. Treatment of gp200 with different glycosidases shows that gp200 is susceptible to N- and O-glycanase but not to endoglycosaminidase H.
• 6.6. On extraction of gp200 with Triton X-114 it partitions exclusively into the detergent-rich fraction consistent with being an integral membrane protein.

     

Comparison of fibrinogenases isolated from copperhead venoms

• 1.1. Fibrinogenase activity from fractions obtained by ion-exchange chromatography of whole copperhead (Agkistrodon contortrix) venoms were further purified by molecular sieve chromatography.
• 2.2. All four enzymes had apparent mol. wt of 23,000–24,000, both non-reduced and reduced as determined by sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography.
• 3.3. All four enzymes rapidly degraded the α-chain of the fibrinogen while apparently leaving the β- and γ-chains intact.