A possible cause of heterogeneity of mammalian apurinic/apyrimidinic endonuclease

• 1.1. Mammalian major apurinic/apyrimidinic (AP) endonuclease, APEX nuclease (M_r 35.4 kDa) was purified from HeLa cells. A hybrid protein (M_r 36.4 kDa), which was expressed in BW2001 strain cells of E. coli, comprising human APEX nuclease headed by 10 additional amino acids was also purified.
• 2.2. The purified preparations were frequently associated with 31-, 33- and 35-kDa peptides having AP endonuclease activity.
• 3.3. The 33- and 35-kDa peptides were suggested to be formed from the hybrid protein or APEX nuclease during their purification processes by proteolytic cleavage with subtilisin-like protease. The 31-kDa peptide was thought to be produced by chemical cleavage of the aspartyl-prolyl bond of APEX nuclease.
• 4.4. The results support the notion that some of AP endonuclease heterogeneity based on the molecular weight difference are caused by proteolytic (and chemical) cleavage of a species of AP endonucleases during the extraction and purification.

     

Purification,characterization and biological activities of phospholipase a from russell's viper (Vipera russelli) venom

• 1.1. The major phospholipase A has been purified to electrophoretic homogeneity from the venom of Vipera russelli (Russell's viper).
• 2.2. The molecular weight of the purified enzyme was estimated to be 31,000 by Sephadex G-75 gel filtration chromatography and 29,000 by SDS-polyacrylamide gel electrophoresis. The enzyme exhibited an apparent K_m value of 2.3 × 10⁻² M.
• 3.3. The phospholipase A showed edema forming, indirect hemolytic and myonecrotic activities but not hemorrhagic activity.

     

Identification of a glycoprotein complex containing a glycogen synthase isozyme in Ascaris Suum obliquely-striated muscle

• 1.1. A glycogen/protein complex which contains the major portion of glycogen synthase activity in Ascaris suum muscle has been purified.
• 2.2. The complex contains two proteins which can be dissociated from a glycoprotein component.
• 3.3. The glycoprotein contains glycogen-like domains and is resistant to trypsin digestion.
• 4.4. The glycogen synthase activity in the purified complex catalyzes glycogen synthesis in the absence of exogenous glycogen, but demonstrates an absolute glucose 6-phosphate requirement for activity.
• 5.5. The data support the hypothesis that this isozyme of glycogen synthase is significantly different from the cyclic AMP-regulated enzyme.

     

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.

     

Purification and some properties of apurinic/apyrimidinic dna endonucleases in rat liver

• 1.1. Three kinds of apurinic/apyrimidinic (AP) DNA endonucleases, APcI, APcII, APcIII were purified from rat liver chromatin.
• 2.2. Molecular weights of APcI, APcII and APcIII were 30,000, 42,000 and 13,000 Da, which have isoelectric points of 7.2, 6.3 and 6.2, respectively.
• 3.3. Mg²⁺ was essential for the activities of these 3 enzymes, and sulfhydryl compounds (βercaptoethanol) had a stimulatory effect on the enzyme activities while N-ethylmaleimide and HgCl₂ inhibited the enzyme activity.
• 4.4. K_m values of APcI, APcII and APcIII for AP site of DNA were 0.53, 0.27 and 0.36 μM, respectively, and AMP was the most potent inhibitor to these three enzymes among nucleotides tested.

     

Purification and partial characterization of an AMP deaminase from the marine invertebrate Palaemon serratus

• 1.1. AMP deaminase from Palaemon serratus tail muscle was partially purified by chromatography on cellulose phosphate.
• 2.2. Muscle homogenates expressed very low enzyme activities and the presence of ATP was necessary to detect AMP deaminase. The specific activity and substrate affinity of the purified enzyme were also very low.
• 3.3. The purified prawn muscle AMP deaminase was contaminated by contractile proteins, one of the major contaminants being actin.
• 4.4. The enzyme displayed a very high affinity for actomyosin which was only partially abolished by pyrophosphate.

     

Domain structure of the 90-kDa stress protein: Heparin- and antibody-binding domain

• 1.1. To understand the physiological roles of the 90-kDa stress protein (HSP90), we investigated the heparin- and antibody-binding domains of the protein.
• 2.2. For heparin-binding sites, HSP90 was digested completely with trypsin, and the digests were applied to a heparin-Sepharose column and eluted with 1.0 M NaCl, followed by 8.0 M urea.
• 3.3. Each elutant was purified by a reverse-phase C₁₈ column.
• 4.4. Two peptides from the NaCl-eluted fraction and no peptide from the urea-eluted fraction were purified.
• 5.5. The purified peptides were sequenced by an automated peptide sequencer.
• 6.6. One of the heparin-binding sites was present between Leu-362 and Arg-365; another was present between Leu-645 and Lys-648.
• 7.7. These two peptides were basic and considerably hydrophilic.
• 8.8. For antibody-binding sites, HSP90 was mildly digested with trypsin, electrophoresed on SDS-polyacrylamide gels and transferred to PVDF membranes.
• 9.9. The four bound of the trypsin fragments could be sequenced with a peptide sequencer.
• 10.10. There was only one antibody-binding peptide, 38 kDa, starting from Pro-2. The others showed no cross-reactivity with the antibody and started from Leu-283.
• 11.11. Therefore, the epitopes of HSP90 are present between Pro-2 and Leu-282.
• 12.12. The heparin-binding sites are present from the middle region of the HSP90 molecule, and the antigen sites are at the N-terminal domain.

     

Mitogenic activity and immunological properties of bolesatine,a lectin isolated from the mushroom Boletus satanas Lenz

• 1.1. A lectin has been purified from the mushroom Boletus satanas Lenz.
• 2.2. The protein, called bolesatine, is mitogenic for human T lymphocytes in a dose- and time-dependent manner.
• 3.3. Optimal mitogenic doses induce the release of interleukin-1α and interleukin-2 from mononuclear cell cultures.

     

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.

     

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 properties of tetralysine endopeptidase from Escherichia coli AJ005

• 1.1. A new tetralysine endopeptidase from Escherichia coli AJ005 has been purified about 135-fold.
• 2.2. The peptidase seems to be specific to tetralysine among lysine homopolymers.
• 3.3. The optimal pH was about 7.5
• 4.4. The activity was inhibited by KCN but not inhibited by soybean trypsin inhibitor.
• 5.5. The apparent K_m value was 2.5 × 1O⁻³ M for tetralysine.

     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.

     

Osmoregulatory responses to abrupt salinity changes in the euryhaline gilthead sea bream (Sparus aurata L.)

• 1.1. Gilthead sea breams (Sparus aurata L.) adapted to sea water (SW, 39‰ salinity) and brackish water (BW, 7‰) were submitted to abrupt osmotic stress by transferring the specimens to 7‰ and 39‰, respectively.
• 2.2. Plasma osmolality, Na,⁺ Cl, ⁻ K, ⁺ Ca, ²⁺ cortisol and glucose were measured before and after the transfers.
• 3.3. The transfer from SW to BW led to transitory hypomineralization and hyperglycemia. In long-term adapted fish cortisol level increased, and osmolality slightly decreased.
• 4.4. Conversely, the transfer from BW to SW provoked transitory hypermineralization. In adapted fish, cortisol levels strongly decreased, and osmolality slightly increased.

     

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.

     

Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

     

Isolation of a hemorrhagic toxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copperhead) and pathogenesis of the hemorrhage induced by the toxin in mice

• 1.1. A hemorrhagic toxin was isolated from the venom of Agkistrodon contortrix laticinctus (Broad-Banded Copperhead) by Sephacryl S-200 HR column chromatography followed by high performance chromatography on Waters DEAE 5PW and protein Pak 125 columns.
• 2.2. Homogeneity was determined by the presence of a single band in acrylamide gel electrophoresis with silver staining.
• 3.3. ACL hemorrhagic toxin I has a molecular weight of about 29,000, is slightly acidic, and is a metalloprotease with activity towards the substrates N,N-dimethylcasein and bovine fibrinogen. Although the toxin is able to hydrolyze fibrinogen in vitro, it does not possess any defibrinogenating activity in vivo whereas the crude venom does show this activity. It has similar cleavage specificities to other snake venom hemorrhagic toxins.
• 4.4. ACL hemorrhagic toxin I causes hemorrhage of rapid onset, present within 5 min of intramuscular injection into mice, and the pathogenesis is one of hemorrhage per rhexis in which capillary endothelial cells are ruptured.

     

Purification and characterization of a blue-colored red-fluorescent protein from the silkworm (Bombyx mori L.) larvae

• 1.1. A red-fluorescent blue protein (P600) was purified from the digestive juice of the silkworm (Bombyx mori L.) larvae raised on mulberry leaves.
• 2.2. The purified protein was electrophoretically homogeneous and showed the absorption maxima at 601.5 nm and 278 nm, and the fluorescence maximum at 621 nm.
• 3.3. The molecular weight was estimated to be 540,000 by gel filtration on Sepharose CL-6B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggests that the protein consists of two heterogeneous polypeptide subunits with a mol. wt of 15,000 and 18,000.
• 4.4. The P600 contains excess acidic amino acid residues over basic groups. The polarity and pI were 45.5% and 4.6, respectively.
• 5.5. The production of H₂O₂ was observed in the presence of P600 upon illumination.

     

Induction of autoantibodies against spermatozoa by injection of allogeneic sperm in the Nile Tilapia,Oreochromis niloticus

• 1.1. The sperm-agglutinating factor (SAF) could be induced in the serum of male Nile tilapias, Oreochromis niloticus, by injection of allogeneic sperm.
• 2.2. Only one class of molecules was demonstrated to be SAF in the serum.
• 3.3. Analysis on purified SAF revealed it to be a tetrameric molecule of IgM with a mol. wt of 760kD.
• 4.4. Cross reaction of the IgM with sperm of other teleost species suggests that sperm-specific surface antigens may be in evolution.

     

Neurohypophysial hormones as molecular evolutionary tracers: investigations on the sturgeons Acipenser stellatus and Acipenser guldenstadti

• 1.1. Neurohypophysial hormones of two sturgeon species, Acipenser stellatus and Acipenser guldenstadti, have been purified through molecular sieving on Bio-Gel P4 and reverse-phase high pressure liquid chromatography on Nucleosil C18 columns.
• 2.2. Arginine vasotocin has been identified in both species by its retention time in partition chromatography, amino acid composition and, in the case of A. stellatus, by amino acid sequencing.
• 3.3. A second peptide has been purified and could be α-deamidated vasotocin.
• 4.4. Another peptide with oxytocic activity, distinct from the known oxytocin-like peptides, seems to be present in very small amounts.