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.

     

Purification and characterization of the d-lactate dehydrogenase from Leuconostoc lactis

• 1.1. The d-lactate dehydrogenase from Leuconostoc lactis has been purified in high yield.
• 2.2.The enzyme is a dimer of subunits of M_r = 39,000 and each subunit contains a single thiol group. The N-terminal residue is methionine.
• 3.3. The amino acid composition has been determined and is typical of that of a soluble globular protein.

     

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.

     

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.

     

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.

     

Ferredoxin-glutamate synthase from Chlamydomonas reinhardii. Prosthetic groups and preliminary studies of mechanism

• 1.1. Ferredoxin-glutamate synthase from the green alga Chlamydomonas reinhardii appears to contain as prosthetic groups, 1 FAD, 1 FMN and 1 |3Fe-xS | cluster, per molecule of M_r = 146,000.
• 2.2. The synthesis of glutamate, catalyzed by this enzyme, proceeds through the formation of an enzyme-bound free radical of flavin semiquinone.
• 3.3. This enzyme may catalyze the ferredoxin-dependent reduction of 2-oxoglutarate, in the absence of glutamine.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Induction of a cadmium-binding protein in a unicellular alga

• 1.1. An inducible Cd-binding compound has been detected in a Cd-treated euryhaline alga, Dunaliella bioculata.
• 2.2. The apparent molecular mass of this heat-stable Cd-binding compound, as determined by gel filtration, was about 10,000.
• 3.3. Anion exchange chromatography (on DEAE Sepharose and Mono Q) and autoradiography of non-denaturing PAGE of the M_r 10,000 fractions revealed the presence of a highly acidic Cd-binding protein which differs on its properties from mammal metallothioneins.

     

The yolk polypeptides of a free-living rhabditid nematode

• 1.1. The yolk proteins of hermaphrodite Dolichorhabditis sp. (Nematode, Rhabditida) are composed of at least three polypeptides: VT1, VT2 and VT3 with molecular masses of 175.2, 107 and 82 kDa respectively.
• 2.2. All three yolk polypeptides make up at least one native protein complex which can be resolved by PAGE.
• 3.3. The yolk proteins are glycosylated and can be isolated by chromatography in Con A-Sepharose.
• 4.4. Partial chymotryptic hydrolysis shows that VT2 in different from its C. elegans homologue, YP115.
• 5.5. The main polypeptides synthesized by whole animals are the yolk components which are actively secreted in the incubation medium.

     

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.

     

Chemical cross-links of proteins by using bifunctional reagents

• 1.1. The chemical identification of spatial arrangements of the subunits in oligomeric proteins is exclusively achieved by the analysis of the reaction products of the protein and bifunctional reagents.
• 2.2. Since the pioneer work of Hartman and Wold (Biochemistry6, 2439–2448, 1967) the bifunctional reagent such as bis-imido-esters was first introduced into protein chemistry.
• 3.3. We have listed the non-cleavable and cleavable bis-imido-esters, the N-hydroxy-succinimido-csters and the aryl azides which once photolyzed, become the highly reactive nitrene intermediates.
• 4.4. Different reagents classified as homo- and hetero-bifunctional reagents are also listed.
• 5.5. The advantages and limits of each group as well as their chemical properties are advanced and extensively discussed.

     

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.

     

Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs

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Highlights

• •Quantitative phosphoproteomics of cells treated with sphingolipid analogs or PP2A inhibitor identify novel protein targets of PP2A.
• •PP2A substrates include several nutrient transporter proteins, GTPase regulators and proteins associated with actin cytoskeletal remodeling.
• •Differential regulation of Akt and Gsk3b account for the difference in vacuolating phenotype observed between SH-BC-893 and C2-ceramide.
• •Dynamic phosphoproteomics enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.

     

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.

     

Heat shock proteins from the lungless salamanders Eurycea bislineata and Desmognathus ochrophaeus

• 1.1. Exposing adult salamanders, Eurycea bislineata and Desmognathus ochrophaeus, to heat shocks of 1 hr at 2 or 5°C below Critical Thermhal Maximum (CTM) resulted in the induction of two heat shock proteins (hsps) of approx. M_r 70,000 and 30,000.
• 2.2. Induction patterns in response to similar heat shocks generally differed between the two species.
• 3.3. The milder heat shocks (5°C below CTM) caused different induction patterns than those from the more severe heat shocks, on a tissue-dependent basis. These results indicate that induction of the two hsps is probably independent.

     

Conodipine-P1-3, the First Phospholipases A2 Characterized from Injected Cone Snail Venom*

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Highlights

• •Three novel Conodipines P1-3 in the injected venom of Conus purpurascens.
• •Conodipines P1-3 have consensus catalytic characteristics of sPLA₂.
• •We determined multiple modification sites in Conodipines P1-3.
• •Evaluated the activity of Conohyal-P1 by a MS-based method.