Expression of lactic dehydrogenase isoenzymes in rabbit muscle during development

• 1.1. Rabbit cDNA probes for H and M lactic dehydrogenase subunits were used to monitor mRNA levels in different muscle types during growth.
• 2.2. At the same time, lactic dehydrogenase activity and relative quantities of H and M protein subunits were measured.
• 3.3. The main results are that mRNA abundance depends on muscle type and age, and mRNA abundance is not correlated with enzymatic activity.

     

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.

     

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 particular kinetic properties of rat spleen cytosolic deoxynucleotidase

• 1.1. Rat spleen cytosolic deoxynucleotidase was purified 40,000-fold to almost homogeneity and had a specific activity of 3000 μmol/min per mg.
• 2.2. Molecular mass of the native enzyme was 45 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the native enzyme comprises two identical 27-kDa subunits.
• 3.3. Specific enzyme activity increases with increasing concentration of enzyme protein and approaches a plateau at high enzyme concentrations.
• 4.4. Enzyme activity increases gradually and nonlinearly with increasing concentration of enzyme in the low concentration range. Above a certain concentration the increase attains a maximal and constant slope.
• 5.5. The kinetic properties can be explained by assuming dissociation of the enzyme into subunits with low or no activity.

     

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.

     

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.

     

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%).

     

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.

     

Investigation of phycocyanin-membrane interaction: Promotion of membrane interactions

• 1.1. In a continuing investigation of phycocyanin-membrane surface interaction, fluorescence quenching experiments were performed with a mixture of two populations of fluorescence probe-encapsulated phospholipid bilayer vesicles in the presence and absence of phycocyanin.
• 2.2. These membrane vesicles were prepared with 1,2-dimyristoyl phosphatidylcholine (DMPC), cholesterol and a probe molecule.
• 3.3. A fluorophore was encapsulated in one population of membrane vesicles, while a quencher was encapsulated in another population of membrane vesicles.
• 4.4. The result was compared with those of experiments in the presence of other biomolecules, including albumin, cytochrome c, hemoglobin, myoglobin or RNA.
• 5.5. Interestingly, a one-third reduction of the fluorescence intensity was observed in the mixture of these two populations of membrane vesicles in phycocyanin's presence.
• 6.6. In contrast, the other biomolecules caused no significant reduction in the fluorescence intensity.
• 7.7. These findings were evidence of a phycocyanin-induced membrane perturbation.
• 8.8. This was further demonstrated by a phycocyanin-induced change in the thermotropic behavior of DMPC vesicles, as measured by differential scanning microcalorimetry.
• 9.9. Such a unique property of phycocyanin is believed to be associated with its known membrane surface-interacting character.
• 10.10. A possible phycocyanin-modulated membrane-membrane interaction was discussed.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Comparative morphological,anatomical and biochemical studies of the urticating apparatus and urticating hairs of some lepidoptera: Thaumetopoea pityocampa Schiff., Th. processionea L. (Lepidoptera,Thaumetopoeidae) and Hylesia metabus Cramer (Lepidoptera,Saturniidae)

• 1.1. Morphological similarities and differences for the urticating apparatus of three Lepidoptera were studied using a scanning electron microscope.
• 2.2. Complementary anatomical studies of the urticant apparatus were undertaken to explain the morphological results.
• 3.3. Biochemical identity of a thaumetopoein-like protein (an urticating protein) was demonstrated for Thaumatopoea urticating hairs but not for Hylesia moth spicules.
• 4.4. Urticating mechanisms appear to be different across species of Lepidoptera.

     

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.

     

SETD1A Methyltransferase Is Physically and Functionally Linked to the DNA Damage Repair Protein RAD18

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Highlights

• •Protein interaction screen of SETD1A/COMPASS complex subunits.
• •Unexpected interaction with DNA damage protein RAD18 was confirmed for SETD1A, but not for other subunits.
• •SETD1A and/or RAD18 influence each other's mRNA and protein expression levels, and disruption of either gene elicits a similar DNA damage sensitivity phenotype.

     

Purification and characterization of haemagglutinin in the haemolymph of the silkworm,Bombyx mori

• 1.1. Changes in haemagglutinating activity in haemolymph during 4th and 5th larval instars of Bombyx mori were stage specific. The activity increased in time concomitant with an increase in the secretory activity of prothoracic glands.
• 2.2. The protein with haemagglutinating activity was purified by ammonium sulfate fractionation, gel-filtration on Sephacryl S-300 and affinity chromatography using either glucuronic acid or galacturonic acid as a ligand.
• 3.3. Western blotting analysis using antibody raised against this protein revealed that Bombyx haemagglutinin is a tetramer composed of two different subunits with mol. wts of ca 88,000 and 90,000.

     

Purification and characterization of a manganese containing superoxide dismutase from bovine heart mitochondria

• 1.1. A manganese containing superoxide dismutase from bovine heart mitochondria was isolated and characterized.
• 2.2. It has a molecular weight of about 86,000 and is composed of 4 noncovalently bound subunits of equal size.
• 3.It appears to contain 2 mole manganese per mole enzyme.
• 4.The carbohydrate content is very low.
• 5.The specific activity and amino acid composition are similar to those of other mitoehondrial superoxide dismutases.
• 6.The enzyme forms complexes with ampholytes and can therefore not be analysed by isoelectric focusing.

     

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.

     

Crystalline style proteins from bivalve molluscs

• 1.1. Proteins from crystalline styles of twelve species of bivalve mollusc were examined under different gel electrophoresis conditions and stained to reveal both protein and carbohydrate.
• 2.2. Native extracts of styles produced relatively few protein bands, however denaturation with SDS resulted in much more complex zymograms.
• 3.3. All species possessed several prominent high mol wt glycoproteins.
• 4.4. Eulamellibranchia all had a major non-glycosylated protein at approx. 62,000 mol. wt.
• 5.5. Most Filibranchia had a major non-glycosylated protein at 37,000–50,000 mol. wt.
• 6.6. Eulamellibranchia were a much more homogeneous group than the Filibranchia.

     

Evidence of presence of a low molecular weight,non-metallothionein-like metal-binding protein in the marine gastropod Nassarius reticulatus L.

• 1.1. A low molecular weight metal-binding protein was found in the snail Nassarius reticulatus cytosol, which was induced in heavy metal contaminated environments.
• 2.2. In our sodium dodecyl sulfate-mercaptoethanol polyacrylamide gel systems it behaved as a protein of 19 kDa mol. wt.
• 3.3. Amino acid composition studies definitely established this protein not to be metallothionein (Mt) like, because it had a much lower level of cysteine and substantial amounts of aromatic amino acids and histidine.
• 4.4. The metal-binding strength of this protein was concluded to be much weaker than that of Mt.
• 5.5. In the crustacean Pagurus bernhardus L. such a protein could not be demonstrated.
• 6.6. In both the snail and the crustacean Zn may inhibit the accumulation of Hg. The premise for studying the induction of the metal-binding Nassarius protein as a supplement to environmental metal monitoring purposes is briefly discussed.

     

Altering erythrocyte membrane composition with phospholipid exchange protein

• 1.1. In establishing a protocol for the use of a phospholipid (PL) 2 exchange protein (PLEP) preparation to incorporate fluorescent lipids into membrane fragments, the potential for the PLEP preparation to alter membrane composition was investigated.
• 2.2. Cholesterol was removed from erythrocyte ghosts and PL content was increased after incubation of the membranes with phosphatidylcholine (PC) vesicles and a nonspecific bovine liver PLEP preparation.
• 3.3. The elevated PL/cholesterol ratio of the ghosts decreased fluorescence polarization of diphenylhexatriene (DPH), indicating an increased average membrane fluidity.
• 4.4. The increased PL/cholesterol ratio and reduced DPH fluorescence polarization were prevented by including cholesterol in the PC vesicles during the initial incubation of ghosts with PLEP preparation and lipid.
• 5.5. In addition, the PC content of ghost membranes was elevated after incubation with PLEP preparation and either PC or mixed PC-cholesterol vesicles.

     

Effects of Acetylation and Phosphorylation on Subunit Interactions in Three Large Eukaryotic Complexes

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Highlights

• •Novel PTMs detected in native yeast exosome, RNA polymerase II and proteasome.
• •MD based approach outperforms published tools in predicting PTMs stability effect.
• •Dynamical approach reveals long range effects of PTMs on subunits binding.
• •Acetylations may have a role in local stabilization of protein complex formation.