Isolation and characterization of the major phospholipase a2 from the venom of trimeresurus purpureomaculatus (shore pit viper)

• 1.1. The major phospholipase A₂ (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity.
• 2.2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14.000 as estimated by SDS-polyacrylamide gel electrophoresis.
• 3.3. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC > PE > PS = 0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme.
• 4.4. The phospholipase A₂ exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 μg/g by i.v. route.

     

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.

     

Improved isolation and further characterization of beta-trichosanthin,a ribosome-inactivating and abortifacient protein from tubers of trichosanthes cucumeroides (cucurbitaceae)

• 1.1. Beta-trichosanthin was isolated from root tubers of Trichosanthes cucumeroides with a procedure involving acetone fractionation, ion exchange chromatography on CM-Sepharose and DEAE-Sepharose and gel filtration on Sephadex G-50.
• 2.2. The protein was homogeneous by SDS-polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. It possessed a molecular weight of 28,000 and was a strongly basic glycoprotein.
• 3.3. It was immunochemically identical to trichosanthin but different from alpha- and beta-momorcharins.
• 4.4. It possessed potent abortifacient and ribosome-inactivating activities. In the latter type of activity it was more potent than trichosanthin.

     

Characterization of a blue astaxanthin protein from the carapace of the crayfish Astacus leptodactylus

• 1.1. A blue carotenoprotein (λ_max = 634 nm) containing astaxanthin as prosthetic group, was extracted and purified from the carapace of the crayfish Astacus leptodactylus.
• 2.2. The blue carotenoprotein contained (3S,3′S)-astaxanthin, (3R,3′S, meso)-astaxanthin and (3R,3′R)-astaxanthin in relative ratio 38:41:21.
• 3.3. The blue carotenoprotein had an approximate mol. wt of 440,000 (gel filtration) and 437,000 (gradient gel electrophoresis).
• 4.4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated the presence of two polypeptides of 19,600 and 18,600 daltons, with different mobility in polyacrylamide gel electrophoresis in the presence of 6 M urea.
• 5.5. At low ionic strength and in the presence of denaturing agents such as SDS, urea, extreme pH and heat, the blue complex showed a greater stability than most of the carotenoproteins studied to date.

     

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm,Bombyx mori

• 1.1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies.
• 2.2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 3.3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm.
• 4.4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.

     

Monoclonal antibodies against rat liver mitochondrial phospholipase a2: Epitope analysis and application in western blotting

• 1.1. Eight cell-lines producing monoclonal antibodies, raised against rat liver mitochondrial phospholipase A₂, were investigated with respect to epitope-recognition. It was shown that all antibodies tested were directed to an identical epitope.
• 2.2. This epitope is a conformational one, since treatment of phospholipase A₂ with the reducing agent dithiothreitol lowered the antibody binding significantly.
• 3.3. To increase sensitivity, Western blot analyses have to be performed on protein samples lacking dithiothreitol or β-mercaptoethanol. The conditions described in this report allow the detection of the phospholipase A₂ in rat liver homogenates.
• 4.4. When rat liver mitochondrial phospholipase A₂ was purified by Ultrogel AcA 54 gelfiltration, a nearly homogeneous protein preparation was obtained, as judged by SDS-PAGE. Western blot analysis of this preparation, however, clearly indicated the phospholipase A₂ to correspond to a hardly visible protein band.

     

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 of endo- and exolaminarinase and partial characterization of the exoacting form from the copepod acartia clausi (Giesbrecht, 1889)

• 1.1. The copepod Acartia clausi exhibited two laminarinases (exo- and endo-acting forms) purified by gel chromatography followed by affinity chromatography. Specific antibodies have been raised against the purified exolaminarinase antigen.
• 2.2. A single band of protein appeared on a polyacrylamide disc gel electrophoresis; its mol. wt is 21,000.
• 3.3. Biochemical properties of the purified enzyme showed a maximum activity at pH 5.2 and a temperature of 40°C with laminarin as substrate. The thermal stability of the enzyme and the effect of various cations on its activity were examined. The enzyme hydrolyses specifically the β(1–3) linked polysaccharides and had no activity against the α(1–4) or β(1–4) disaccharides or polysaccharides.
• 4.4. The kinetic parameters V_m and K_m vary with the temperature; the affinity constant (K_a) was maximum between 25–30°C. The Arrhenius plot defined two values of energy of activation: 7980 cal/mole and 17,506 cal/mole.
• 5.5. From the purification scheme the exoacting form appears to be largely dominant over the endoacting form.

     

Vitellogenesis in the stable fly,stomoxys calcitrans

• 1.1. No female specific proteins were found in the stable fly hemolymph by polyacrylamide gel electrophoresis.
• 2.2. Six major yolk polypeptides (YP1, YP2, YP3, YP4, YP5 and YP6) have been identified in the stable fly. Their mol. wt as determined by SDS-polyacrylamide gel electrophoresis are 41,100, 42,600, 44,100, 46,600, 48,900 and 50,600, respectively.
• 3.3. YP3 was purified and antibody made against it. By using the antibody and in vitro organ culture the stable fly yolk polypeptides were shown to be synthesized exclusively by the ovaries and not the fat body.
• 4.4. The stable fly yolk polypeptides are immunologically similar to yolk proteins of other related flies.

     

Age-dependent proteins in eyes of annual killifishes Pterolebias longipinnis detected by 2-dimensional electrophoresis

• 1.1. Eye proteins of Pterolebias longipinnis have been analyzed by 2-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis during aging from adolescence until normal death.
• 2.2. The protein pattern on the gels changed gradually with progressing age.
• 3.3. In senescent eyes, three protein spots appeared for a time and 36 disappeared from the pattern.
• 4.4. The isoelectric points of the proteins in the presence of urea and the molecular weights in an unreducing buffer are presented.

     

Purification and characterization of a novel chymotrypsin inhibitor controlled by the chymotrypsin inhibitor A (Ict-A) gene from the larval hemolymph of the silkworm,Bombyx mori

• 1.1. On polyacrylamide gel electrophoresis, chymotrypsin inhibitors in the larval hemolymph of the silkworm were found as 15 electrophoretically distinct bands.
• 2.2. One of them, CI-13 migrating fastest toward the anode, was purified from the hemolymph.
• 3.3. The inhibitor was a monomeric protein with a molecular mass of 14,000 and showed stability for both heat and a wide range of pH. The isoelectric point was pH 4.1.
• 4.4. CI-13 was able to inhibit chymotrypsin completely and trypsin slightly, but was ineffective against other proteinases such as papain, ficin, carboxypeptidase A, V8 proteinase, serratia peptidase, cocoonase and proteinases derived from gut juice of the silkworm.

     

Protein characterization of sexually mature and immature forms of Schistosoma mansoni

• 1.1. Protein composition of different stages of Schistosoma mansoni was compared using specific antisera, 2D polyacrylamide gel electrophoresis and 14-C-leucine incorporation into proteins.
• 2.2. Major qualitative differences were detected when an anti-membrane antiserum was used.
• 3.3. 2D gel electrophoresis showed that the protein composition varied when mature and immature females were compared, whereas no differences were noted when mature and immature male worms were compared.
• 4.4. Experiments measuring protein synthesis by the different schistosome stages confirmed that upon maturation, only the female schistosomes displayed qualitative differences.
• 5.5. The protein pattern of the male schistosomes did not vary significantly as a function of development.

     

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.

     

Lysosomal acid deoxyribonuclease from vervet monkey livers—I: Purification and physico-chemical characterization

• 1.1. Acid DNase from monkey liver lysosomes was purified to homogeneity by salt extraction of lysosomal membranes at pH 3.8; (NH₄)₂SO₄ fractionation; low salt precipitation; SP-C₅₀ and G-150 Sephadex chromatography; and polyacrylamide gel electrophoresis.
• 2.2. The pH for optimum activity was dual in character with a labile optimum at pH 3.8 and a less active but stable one at pH 4.2
• 3.3. The estimated molecular weight was 40 K and the pl was 4.4.
• 4.4. Inorganic ions such as Ca²⁺, Mg²⁺, Mn²⁺ and SO₄²⁻ were more than 80% inhibitory at 10-mM levels. Fe³⁺ ions were 80% inhibitory at 0.1-mM levels. 15. Nad at 100 mM is essential for activity but becomes 100% inhibitory above 200 mM.

     

Purification of the flavin-containing monooxygenase from mouse and pig liver microsomes

• 1.1. The microsomal flavin-containing monooxygenase has been purified from mouse and pig liver utilizing Cibacron-Blue Sepharose, Procion-Red agarose, and 2'5'-ADP Sepharose.
• 2.2. The enzymes had a final specific activity of 1200 and 954 nmol/min/mg protein from mouse and pig liver respectively.
• 3.3. The enzyme from both mouse and pig liver displayed typical flavoprotein spectra and appeared homogeneous by denaturing polyacrylamide gel electrophoresis.

     

Influence of Carbicron (O-[(2-butenoic acid)-N,N-dimethylamide-3-YL] O,O-dimethylphosphate) on some biochemical and biophysical parameters of rat liver membranes

• 1.1. Treatment of rats with carbicron induced a reduction of the phospholipids in both microsomal and plasma membranes.
• 2.2. A decrease of the structural order parameter (S_DPH) and an increase of the pyrene excimer-to-monomer fluorescence ratio (I_E/I_M) was also observed, indicating membrane fluidization.
• 3.3. The specific activity of membrane-bound phospholipase A₂ and phospholipase C were decreased in both types of membranes, whereas acyl-CoA:lysophosphatidylcholine acyltransferase activity was augmented due to carbicron treatment.

     

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.

     

The molecular weights of Arthropod hemocyanin subunits: Influence of tris buffer in SDS-PAGE estimations

• 1.1. Available molecular weights data for Arthropod hemocyanin subunits as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate are analyzed.
• 2.2. Relationship between buffer composition and subunit mobility in SDS-PAGE is shown by studying Cancer pagurus hemocyanin.
• 3.3. Tris buffers are suspected to give erroneous molecular weight estimations for Arthropod hemocyanin subunits.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Is the metal binding protein metallothionein present in the coelenterate Hydra attenuata?

• 1.1. Studies have been performed on untreated and heavy metal treated Hydra attenuata in order to reveal the presence of low mol. wt metal-binding proteins.
• 2.2. A prepared rat metallothionein (Mt) standard, gel permeation, polyacrylamide gel electrophoresis and autoradiographic techniques were used in the experiments.
• 3.3. Our results indicate that H. attenuata, and three species of marine coelenterates, lack metallothionein (Mt) or other metal binding proteins.