Expression of a putative catalytic domain of the human apex nuclease (a major apurinic/apyrimidinic endonuclease) in Escherichia coli

• 1.1. Sequence analyses of APEX nuclease, a mammalian major apurinic/apyrimidinic (AP) endonuclease homologous to Escherichia coli exonuclease III, suggested that APEX nuclease is organized into two domains, a Mr 6000 N-terminal domain containing nuclear location signals and a Mr 29,000 C-terminal catalytic domain.
• 2.2. In order to study the enzyme structure further, vectors expressing APEX nuclease (pTAPXH1) and the Mr 29,000 C-terminal region (pTAPXH61) were constructed using cDNA (APX cDNA) for the human APEX nuclease and pTrc99A plasmid. The constructs were introduced into BW2001 strain (xth-11, nfo-2) cells of E. coli to produce transformants designated as BW2001/pTAPXH1 and BW2001/pTAPXH61, respectively. Both the APEX nuclease expressed in BW2001/pTAPXH1 and the Mr 29,000 C-terminal peptide expressed in BW2001/pTAPXH61 were partially purified by column chromatography and highly purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 3.3. The purified APEX nuclease and the Mr 29,000 C-terminal peptide both showed equally high AP endonuclease activity which indicates that the Mr 29,000 C-terminal region of the APEX nuclease is (or contains) the AP endonuclease domain.

     

Isolation and characterization of glutamine synthetase from the diazotroph Azospirillum brasilense

• 1.1. Glutamine synthetase was purified from the diazotroph Azospirillum brasilense.
• 2.2. The holoenzyme with a M_r of 630,000 is composed of 12 subunits of M_r 52,000.
• 3.3. A modified subunit of M_r 53,000 was also found by electrophoresis under denaturing conditions.
• 4.4. It is shown that the M_r 53,000 species is the adenylylated subunit.
• 5.5. The apparent K_m values for glutamate, ATP and ammonia were 2.5 ± 0.3 mM, 200 ± 20 μM and42 ± 2 μM, respectively.
• 6.6. Levels of glutamine synthetase activity in A. brasilense cells varied by a factor of 8 depending on the nitrogen source and its concentration in the growth medium.

     

Detection of 180 kDa proteins in electroplax sodium channel preparations

• 1.1. Co-isolating proteins (M_r 170,000–220,000) from sodium channel preparations made from the electric organ of the electric eel (Electrophorus electricus) were detected on Western blots using monoclonal a antibodies.
• 2.2. Similar protein patterns were seen on immunoblots containing immunoprecipitated protein from eel muscle and brain tissues but not heart.
• 3.3. These co-isolating proteins could be separated from the mature TTX-sensitive channel protein (M_r 280,000) using a lentil lectin-Sepharose column.
• 4.4. The 180 kDa proteins do not appear to be channel-related and can be detected as contaminants in electroplax sodium channel preparations using the monoclonal antibodies described here.

     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

Purification and properties of aldehyde dehydrogenase from Aspergillus nidulans

• 1.1. Aspergillus nidulans produces aldehyde dehydrogenase (ALD-DH) only when grown in the presence of ethanol, threonine or acetoacetic acid as inducer. Enzyme formation is inhibited by glucose in the growth medium.
• 2.2. ALD-DH is purified by a rapid procedure using Cibacron Blue Affinity Chromatography with specific inhibitoe elution by NAD plus 2:2′ dithiodipyridine or 2:4 disulfiram.
• 3.3. The pure native enzyme has a M_r=265,000 and a subunit M_r of 540,000. Its optimum pH is 8.5; its preferred substrate is acetaldehyde and it can use either NAD or NADP.

     

Physicochemical property of bovine brain 73-kDa stress protein

• 1.1. An approximately 70-kDa protein was purified from bovine brain using an ATP-Sepharose column.
• 2.2. The protein sample was found to contain two proteins (major 73 kDa and minor 72 kDa) on two-dimensional gel electrophoresis.
• 3.3. Antibodies raised against the 73- and 72-kDa proteins cross-reacted with stress-induced HSP73 and HSP72 from HeLa cells, respectively.
• 4.4. Heparin-binding peptides were obtained from trypsin digests of HSP73.

     

Isolation and properties of carboxylesterases of the termite gut-associated fungus,Xylaria nigripes. k., and their identity from the host termite,Odentotermes horni. w., mid-gut carboxylesterases

• 1.1. The termite, Odentoiermes horni. W., houses three fungal species, viz. Xylaria nigripes, Termitomyces microcorpus, and Trichoderma (species not identified), in its gut. X. nigripes was found to possess higher esterase activity levels than the other two.
• 2.2. Four esterase enzymes, viz. FE-I, -II, -III and -IV, with pI values 5.1, 5.25, 5.4 and 5.6, respectively, were identified, isolated and purified to apparent homogeneity from the fungus X. nigripes, their biochemical and enzymological properties were determined, and compared with those of the previously characterized host termite mid-gut enzymes, TE-I and -II.
• 3.3. The M_r, ofFE-I and -II was 85.1 kDa and those of FE-III and -IV was 87.5 kDa. However, TE-I and -II were relatively smaller (M_r ~ 78.5 kDa). Each of the fungal enzymes, viz. FE-I to -IV, was a homodimer with subunits associated non-covalently. The subunit M_r, were 42.6 kDa for FE-I and -II, and 43.7 kDa for FE-III and -IV. On the other hand, the termite mid-gut enzymes, TE-I and -II, were also homodimeric, but the subunits were associated covalently (subunit M, = 40 kDa). Immunologically the fungal esterase enzymes, viz. FE-I to -IV, were different from those of the host termite mid-gut esterases, viz. TE-I and -II.
• 4.4. The substrate specificity and inhibitor sensitivity studies classify these enzymes, i.e. FE-I to -IV, as carboxylesterases (EC 3.1.1.1). Steady-state product inhibition kinetics suggested; an ordered release of products, i.e. alcohol followed by acid, and a Uni-Bi kinetic reaction mechanism.
• 5.5. The two preliminary studies, i.e. the confinement of most esterase activity to the gut-tissue free from microorganisms and starvation of termites not leading to complete loss of esterase activity in the gut of the termites, suggested that there may not be any symbiotic relationship between termite, O. horni, and its gut associated microorganisms with regard to ester metabolism. Though the enzymes from the two sources were carboxylesterases, several of their properties were different and hence, they are different entities.

     

The effect of γ-radiation on the NADP-MALIC enzyme from mango fruit,mangifera indica—I. Physico-chemical aspects

• 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in dilute solution and the effect of γ-irradiation was investigated.
• 2.2. The activity of the enzyme decreased exponentially as a function of the applied dose under all conditions investigated. The inactivation yield (Go-value) in neutral solution and in air was 0.069.
• 3.3. The role of the radicals produced by water radiolysis in the inactivation of the enzyme was investigated by using different gas atmospheres and selective free radical-anions. The hydrogen atom and the hydrated electron (reducing species) were found to be important in the enzyme inactivation; as well as the possible destruction of cysteine and tryptophan residues.
• 4.4. The irradiated enzyme appears to adopt a more compact conformation as reflected in a slightly lower M_r, Stokes-radius and diffusion coefficient.
• 5.5. γ-Radiation does not lead to any heterogeneity in the charge and size properties of the enzyme and the pI and the M_r of the subunits were unaffected.
• 6.6. Some differences in the amino acid composition of the non-irradiated and irradiated enzyme were observed but specific amino acid residues were not preferentially destroyed.
• 7.7. These changes were also reflected in the ultraviolet spectrum of the enzyme which shifted to lower values.
• 8.8. The major cause of inactivation seem to be a change in conformation caused by chemical modification of amino acid side chains.

     

Degradation of bone matrix proteins by osteoclast cathepsins

• 1.1. The degradation of the bone matrix proteins osteocalcin, osteonectin and α₂HS-glycoprotein by human cathepsins B and L and human osteoclastoma cathepsins has been investigated.
• 2.2. Intermediate degradation products (M_r > 12kDa) were not observed during the digestion of α₂HS-glycoprotein and osteonectin by cathepsins B and L although they were observed with some of the osteoclastoma cathepsins. Most of the osteoclastoma cathepsins were capable of degrading these two proteins to small peptides at comparable rates.
• 3.3. Each cathepsin produced a different pattern of osteocalcin degradation products.
• 4.4. The extensive range of non-collagenous proteins in bone matrix may necessitate the production by osteoclasts of cathepsins with different specificities during bone resorption.

     

Tissue specific isoenzymes of d-lactate dehydrogenase from the foot,mantle and hepatopancreas of Patella caerulea (L). purification and properties

• 1.1. Isoenzymes of d-lactate specific dehydrogenase from foot, mantle and hepatopancreas of Patella caerulea have been purified by Chromatographic techniques. d-lactate dehydrogenase (d-Ldh) from P. caerulea tissues was found to be tetrameric with a M_r of ca 140,000 as judged by gel filtration; subunit M_r of ca 37,000 was obtained from SDS-electrophoresis.
• 2.2. Kinetic studies suggest that P. caerulea foot and mantle d-Ldh is similar to vertebrate muscle-type l-Ldh; furthermore hepatopancreas d-LDH resembles vertebrate heart-type l-LDH since it has a higher affinity for d-lactate and is inhibited by pyruvate.
• 3.3. The results imply that the P. caerulead-Ldh isoenzymes may have distinct metabolic functions.

     

A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline

• 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-P_i basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
• 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
• 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
• 4.4. The method could also be applied to purify PLC produced in a low-P_i complex medium. The resultant preparation was not homogeneous.
• 5.5. The molecular weight for both PLC preparations was about 70 kDa.
• 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent K_M of 25 mM for p-nitrophenylphosphorylcholine.
• 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
• 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP_l-BSM plus choline but not the enzyme from the LP_l-CM.

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.

     

Molecular and spectroscopic characterisation of a low molecular weight seed storage protein from yellow mustard (Sinapis alba L.)

• 1.1. A 1.7S protein has been purified from mustard seeds (Sinapis alba L.). This protein, soluble in water and dilute salt solutions, is considered as an albumin and constitutes about 10% of the total soluble protein in mustard seeds.
• 2.2. Its molecular weight is approximately 15,000 and is composed of two polypeptide chains (M_r = 9500 and 5000), linked by two disulfide bridges.
• 3.3. The amino acid compositions of both subunits as well as of the native protein are reported, showing a strong homology with napins from Brassica napus L.
• 4.4. The ultraviolet absorption, fluorescence emission and circular dichroism spectra of the purified protein have been obtained. The mustard protein exhibits about 50% α-helix with a very low β-structure content. Based on its structural characteristics, a zein-like packing is proposed for this protein from mustard seeds.

     

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.

     

Heterosynthetic origin of the major yolk protein,vitellin, in a nereid,Perinereis cultrifera (polychaete annelid)

• 1.1. An examination of proteins synthesized by Perinereis cultrifera oocytes incubated in vitro with [³H]leucine clearly shows that these cells are not capable of synthesizing the main yolk protein previously identified in this worm.
• 2.2. In addition, the detection of radiolabelled vitellin in oocytes after in vitro incubation of an oocyte-coelomocyte cell mixture in presence of [³H]leucine strongly suggests that the coelomocytes, free cells in the coelomic cavity, synthesize and secrete a vitellin precursor, vitellogenin, that is subsequently taken up by the oocytes.
• 3.3. Two native proteins differing in mol. wt but reacting with anti-vitellin antibodies have been identified in coelomocyte incubation medium. Also found in the coelomic fluid, they have been designated VG1 (M_r = 530,000) and VG2 (M_r = 320,000).
• 4.4. The two vitellogenins consist of a single type of polypeptide of M_r = 176,000 and are incorporated in the oocytes where they are apparently observed under a single molecular form corresponding to VG1, the highest mol. wt protein similar in size to the initial form of vitellin (VI, 530,000).
• 5.5. From these data, it seems likely that VG2 is a monomeric molecule that is taken up by the oocytes as a dimer of VG1.
• 6.6. We conclude that P. cultrifera accumulates vitellin heterosynthetically and that vitellogenin is produced by the coelomocytes. Moreover, a single polypeptide similar in size to the polypeptidic component of secreted vitellogenin has been detected in the coelomocytes.
• 7.7. Since this polypeptide has been identified previously as the single intraoocytic precursor of the four lower mol. wt products that make up the mature form of vitellin (V5), it appears that P. cultrifera exhibits for vitellogenin a processing pathway in which cleavage of the precursor occurs only after uptake by the oocyte.

     

A lipid transfer particle in Musca domestica haemolymph

• 1.1. In Musca domestica haemolymph a lipid transfer particle (LTP) is present.
• 2.2. Musca domestica LTP is able to catalyze the transfer of lipids between different housefly lipophorin forms and also between lipophorins of Diptera and Lepidoptera.
• 3.3. The lipophorin of larval Dione juno (Lepidoptera) was purified and is composed of two apolipoproteins, apolipophorin I (M_{r = 209,000}) and apolipophorin II (M_{r = 85,000}) with a density of 1.124 g/ml.
• 4.4. The density of housefly lipophorin undergoes variations during the gonotrophic cycle.
• 5.5. The lipophorin density variation results suggest that when a high rate of lipid utilization occurs, the lipophorin has a higher density value.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

Purification and properties of an ld-dipeptidase FROM Bacillus sphaericus

• 1.1. An ld-dipeptidase (EC 3.4.13.-) that hydrolyzes the unrelated dipeptides l-Ala-d-Glu (sp. act. 0.85 μmol·min⁻¹·mg⁻¹) and l-Lys-d-Ala (sp. act. 11 μmol · min⁻¹·mg⁻¹) has been purified 250-fold from the sporulation medium of Bacillus sphaericus with a 4% recovery of lytic activity.
• 2.2. Throughout the purification steps, followed with both substrates, the enzyme peaks of activities were congruent and the ratios of activities were constant. Both activities were activated 50-fold by cobalt. Polyacrylamide gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. The data are consistent with those activities being due to a single enzyme.
• 3.3. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band (M_r 38,000).
• 4.4. This dipeptidase hydrolyzes some other ld-dipeptides with a free amino and carboxyl group. Although dipeptides having a di-amino acid as the amino terminus are the best of the substrates tested, the hydrolysis occurs also when neutral amino acids are N-terminal. The activity is higher with neutral C-terminal residues such as Gly or d-Ala than with a di-acid residue such as d-Glu.
• 5.5. This enzyme may have a function in peptidoglycan metabolism.

     

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%).

     

Partial characterization and subunit analysis of major phosphoproteins of egg yolk in the fish,Oryzias latipes

• 1.1. Molecular weight estimation and subunit analysis of four yolk phosphoproteins (PP1-PP4) in medaka (Oryzias latipes) eggs were performed.
• 2.2. PP1 (M_r ≈ 210,000) and PP2 (M_r ≈ 180,000) were found to be heterodimers composed of subunits of 113,000 and 94,000 and subunits of 84,000 and 72,000, respectively.
• 3.3. PP3 and PP4 [phosvitins of medaka (Murakami et al., 1990, Devl. Growth Differ.32, 619–627)], were monomeric phosphoproteins having mol. wts of about 40,000 and about 20,000, respectively.
• 4.4. Lipid composition of the mixture of PP1 and PP2, vitellogenin and yolk were found to be almost the same. PP1 and PP2 are probably lipovitellins of medaka.