The interaction of dodecyl and tetradecyl sulfate with proteins during polyacrylamide gel electrophoresis.

1. The affinity of tetradecyl sulfate for many unfolded proteins is greater than that of dodecyl sulfate. 2. It is the presence of tetradecyl sulfate that results in the staining of proteins by pinacryptol yellow seen by Stoklosa and Latz (Stoklosa, J.T. and Latz, H.W. (1974) Biochem. Biophys. Res. Commun. 58, 74--79), as some tetradecyl sulfate remains associated with proteins during electrophoresis at room temperature (as opposed to dodecyl sulfate which, within the limit of detection, is completely removed). 3. Tetradecyl sulfate has a greater capacity to dissociate protein aggregates which consist of identical peptide chains, such as Glycophorin dimers and bovine serum albumin dimers, than does dodecyl sulfate.     

Unfolding and release of heme from human hemoglobins A,S and F

• 1.1. Analysis of the Soret spectra of hemoglobins A, S and F has been used to determine the extent of heme exposure and release from these hemoglobins in the presence of several solvent perturbants.
• 2.2. Oxyhemoglobin S unfolding in the presence of either urea or propyl urea resulted in greater heme exposure and release than either oxyhemoglobins A or F.
• 3.3. Methemoglobin formation resulted in lower denaturation midpoints for each hemoglobin compared to the reduced oxyhemoglobin state; methemoglobin F had the lowest denaturation midpoint under isothermal denaturing conditions.
• 4.4. Rate of heme exposure was greater for oxyhemoglobin S than oxyhemoglobin A in the presence of 200 μM the anionic detergent sodium dodecyl sulfate.
• 5.5. Evidence for increased levels of heme release in hemoglobin S may be related to the greater tendency of sickled red cell membranes to undergo lipid oxidation.

     

Subunits of Potamilla leptochaeta chlorocruorin

The dissociation of the extracellular chlorocruorin of Potamilla leptochaeta was investigated by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS).

• 1.1. The unreduced chlorocruorin dissociated into three subunits with molecular masses of 15,000 (1), 30,000 (2) and 65,000 (3); reduced chlorocruorin provided one broad band of molecular mass 14,000 ± 2000.
• 2.2. Reelectrophoresis of each of the three subunits in the presence of 2-mercaptoethanol provided one broad band having a molecular mass of about 13,000 ± 2000.
• 3.3. It is proposed that Potamilla chlorocruorin consists of interchain disulfide-bonded dimers and tetramers of polypeptide chains of ca. 14,000 ± 2000.
• 4.4. Comparison of the subunit relationships observed in annelid extracellular hemoglobins with those seen in chlorocruorins suggests that there are differences between the two molecules, insofar as aggregation of the smallest subunit is concerned: although it associates into dimers and trimers in hemoglobins, it forms dimers and tetramers in chlorocruorins.

     

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.

     

Monoclonal antibodies prepared against heparin lyase I and their reactivity toward heparin lyase I,II and III

• 1.1. Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared.
• 2.2. The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting.
• 3.3. Individual antibodies showed different reactivity toward the three heparin lyases.
• 4.4. The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate.
• 5.5. The antibodies can be used to rapidly distinguish between the three heparin lyases.

     

Characterization of spore coat proteins of Bacillus thuringiensis and Bacillus cereus

• 1.1. Spore coat extracts from Bacillus thuringiensis subspecies kurstaki and israelensis and Bacillus cereus T and B. cereus NRRL 569 were characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by amino acid analysis.
• 2.2. Both B. cereus spore coats had similar electrophoretic profiles.
• 3.3. The B. thuringiensis spore coats contained crystal proteins as major components as well as lower mol. wt proteins.
• 4.4. B. thuringiensis subsp. israelensis had a unique coat protein profile which was different from B. cereus and B. thuringiensis subsp. kurstaki coats.
• 5.5. Insecticidal activity of spores against the tobacco hornworm, Manduca sexta, and the mosquito, Aedes aegypti, also was determined.
• 6.6. B. thuringiensis subsp. kurstaki spores were lethally toxic to the tobacco hornworm (Lepidoptera) larvae, whereas spores of the other subspecies were not.
• 7.7. Except for subspecies israelensis, none of the spores was effective against the mosquito (Diptera) larvae.

     

Comparison of fibrinogenases isolated from copperhead venoms

• 1.1. Fibrinogenase activity from fractions obtained by ion-exchange chromatography of whole copperhead (Agkistrodon contortrix) venoms were further purified by molecular sieve chromatography.
• 2.2. All four enzymes had apparent mol. wt of 23,000–24,000, both non-reduced and reduced as determined by sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography.
• 3.3. All four enzymes rapidly degraded the α-chain of the fibrinogen while apparently leaving the β- and γ-chains intact.

     

Electrical transport characteristics of Aplysia californica intestinal epithelium

• 1.1. Replacing chloride (Cl⁻) with sulfate (SO₄²⁻) in the bathing medium drastically reduced the mucosal membrane potential difference (ψ_m).
• 2.2. The voltage divider ratio was significantly greater than one.
• 3.3. Mucosal ^d-glucose decreased the input resistance of the intestinal epithelium.
• 4.4. Addition of furosemide to the mucosal bathing medium inhibited transepithelial potential difference and short-circuit current.
• 5.5. Addition of SITS to the mucosal bathing medium partially inhibited transepithelial potential difference and short-circuit current.
• 6.6. Diffusion potentials in the intestinal epithelium were symmetrical.

     

Prostaglandin E1 and analogs of prostacyclin influencing superoxide production by the human neutrophil nadph oxidase system

• 1.1. The effects of prostaglandin (PG) E₁, and I₂ analogs (OP-41483 and OP-2507) on the Superoxide generation of human neutrophil NADPH oxidase (EC 1.6.99.6) in both whole-cell and cell-free systems were investigated.
• 2.2. In a whole-cell system, OP-2507 inhibited the Superoxide generation by neutrophils exposed to phorbol myristate acetate concentration-dependently through its superoxide-scavenging action.
• 3.3. The concentration of the drug required for 50% inhibition of the oxidase (IC₅₀) was 21 μM.
• 4.4. In a cell-free system, however, the drug in concentrations of < 100 μM did not inhibit the activation of NADPH oxidase by sodium dodecyl sulfate because of its inactivation by the detergent.
• 5.5. Although PGE₁ and OP-41483 did not inhibit the Superoxide production by stimulated neutrophils in a whole-cell system, they both inhibited the activation of NADPH oxidase in a cell-free system concentration-dependently, with IC₅₀ values of 44 and 170 μM, respectively.
• 6.6. In addition, in the cell-free system, the K_m value for NADPH of the oxidase was unchanged by PGE₁.
• 7.7. The results suggest that the PGI₂ analog, OP-2507, is a possible superoxide-scavenger and that PGE₁ inhibits the NADPH oxidase activation by sodium dodecyl sulfate in a cell-free system concentration-dependently.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

A comparative study of vertebrate eye lens crystallins using isoelectric focusing and densitometry

• 1.1. The crystallin proteins of numerous species belonging to different classes of vertebrates have been studied.
• 2.2. Species-specific crystallin patterns are revealed which unequivocally characterize the different species.
• 3.3. A marked variability in the number and percentage of alpha-, beta- and gamma-crystallins were found in the various species.
• 4.4. The gamma-crystallin family, with a meagre number of common bands, has proved to be most representative of the species. The beta-crystallins, with their greater number of common bands, have been best preserved throughout vertebrate evolution.
• 5.5. From the similarity coefficient matrix a dendrogram is drawn up, a visual phylogenetic summary of the interrelationships between the vertebrates considered.
• 6.6. In the Discussion, other aspects are considered, such as lens morphology, functionality, animal age, post-synthetic modifications and genetic factors.

     

Isolation and characterization of new minor proteins (Ovoglycocomponents) from chicken and quail egg whites

• 1.1. New minor proteins were isolated from chicken and quail egg whites and were named the ovoglycocomponent.
• 2.2. They migrated on polyacrylamide-gel electrophoresis as a single band without any contaminations.
• 3.3. They contain a considerable amount of carbohydrates, of which hexosamine was much higher in the chicken than in the quail.
• 4.4. The molecular weights of the chicken and quail ovoglycocomponents estimated from gel filtration were approx 56,000 and 49,000, respectively.
• 5.5. The isoelectric points were measured as 5.1 for the chicken and 5.4 for the quail.

     

Effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail

• 1.1. The effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail were investigated.
• 2.2. The level of liver NAD was not affected by niacin deficiency whereas the level of pectoral muscle NAD was markedly reduced.
• 3.3. In all dietary treatments the liver had the highest turnover rates of proteins, heart and brain had intermediate rates, and pectoral muscle had the lowest rates.
• 4.4. Relative turnover rates of proteins in all tissues (particularly pectoral muscle) of the niacin deficient group were significantly higher than those of pair-fed control group, although there were no significant differences in turnover rate between pair-fed control and control groups.
• 5.5. The high turnover rate of proteins in niacin deficiency was primarily attributed to enhanced degradation rate of proteins rather than enhanced synthesis rate of proteins.
• 6.6. Optical density scanning (or densitometric) of water-soluble pectoral muscle proteins separated by isoelectric focusing revealed several additional minor protein bands between major protein bands in the niacin deficient group which were more pronounced in the acidic region of the gel.
• 7.7. These results suggest that proteins with a low pI value in pectoral muscle of the niacin deficient animal are highly sensitive to protein degradation.

     

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.

     

The respiratory pigment of the serpulid polychaete,Serpula vermicularis L. Structure of its chlorocruorin and hemoglobin (erythrocruorin)

• 1.1. The respiratory pigment found in the vascular fluid of Serpula vermicularis appears to be homogeneous with respect to molecular weight (3 × 10⁶), charge (pl = 5.1) and high pH dissociation properties.
• 2.2. Serpula pigment contains one iron per 24,700 g protein with 60% of the iron present as chlorocruoroheme and 40% as heme. This ratio of chlorocruoroheme to heme was found in worms ranging in weight from about 50 to 500 mg.
• 3.3. The pigment is composed of at least three subunits with molecular weights in sodium dodecyl sulfate (SDS) of 13,300, 14,700 and 17,500. The amino acid composition of this pigment is similar to those of other annelid extracellular hemoglobins and chlorocruorins.
• 4.4. Preliminary oxygen equilibrium experiments show that the heme oxygen binding site may combine with oxygen in a manner different from the chlorocruoroheme oxygen binding site.
• 5.5. These results are discussed with respect to possible structures of S. vermicularis pigment, its relationships to other annelid pigments and its function.

     

Allopurinol and theophylline: Their effect on longevity,fertility and behavior of hornets kept under different light and crowding conditions

• 1.1. The present work deals with the effect of feeding hornets on moderate amounts of theophylline and allopurinol.
• 2.2. The median survival time of hornets fed on allopurinol was greater than that of hornets fed on theophylline, while that of a control group fed on sugar solution alone was between the two drug-fed groups.
• 3.3. The controlled-diet hornets, which were kept in the groups of 10, lived longer than hornets in groups of 20 or 40.
• 4.4. Those fed on theophylline usually did not eat proteins, niether did they build cells, lay eggs nor engage in any social activity. They behaved rather like queen hornets during the winter diapause.
• 5.5. Hornets fed on allopurinol built less cells and combs than did the controls. They were very aggressive, produced fewer eggs, attended less brood than did the controls and fed intensively on proteins (partly through cannibalism).

     

Online Parallel Accumulation–Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer

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Highlights

• •Online PASEF achieves greater than 100 MS/MS per second at full sensitivity.
• •Accurate label-free quantification of over 6000 proteins in 2 h.
• •High throughput demonstrated on 50 ng digests measured in 5 min.
• •High-precision determination of 100,000 peptide collisional cross sections.

     

Identification of the major annexins in Ehrlich ascites tumor cells

• 1.1. Three calcium-binding proteins have been purified from Ehrlich ascites tumor cells.
• 2.2. They were identified by amino acid sequence analysis on selected fragments obtained by tryptic digestion.
• 3.3. The proteins belong to the annexin family and were identified as annexins II, III and V.
• 4.4. Antibodies raised against the proteins were used to examine for their presence in a number of murine tissues.
• 5.5. The occurrence was found to be in reasonable accordance with earlier reports.

     

Effect of conjugation on the incorporation of glycine into Tetrahymena

• 1.1. Conjugation of Tetrahymena enhanced the incorporation of glycine into the nuclear fraction by 500%.
• 2.2. Incorporation of glycine into the microsomal supernatant was augmented by almost 500% by conjugation.
• 3.3. Mitochondrial incorporation was stimulated nearly 3-fold in the conjugating strains while the incorporation of glycine into the microsomes was enhanced approximately 2.5 times.
• 4.4. In the whole cell, glycine incorporation was increased nearly 2-fold by conjugation.
• 5.5. Strong nuclear involvement was indicated by elevated metabolic activity and incorporation of glycine into RNA and DNA.
• 6.6. Stimulation of the metabolism of Tetrahymena by cell communication suggests that the contents of a cell can have a synergistic effect on another cell.
• 7.7. Augmentation of the biosynthetic capacities of cells by fusion is a demonstration of the dominant role of the cell membrane in the regulation and control of cells.
• 8.8. Enhancement of biosynthesis of nuclear proteins in conjugating strains of cells indicates that fusion gives rise to the synthesis of new protein from previously existing protein or protein procursors.
• 9.9. The specific activities of the subcellular fractions after the incorporation of glycine into 2 separated starved strains of Tetrahymena followed the usual pattern of nucleus less than whole cells, whole cells less than mitochondria, mitochondria less than microsomes, but with the microsomal supernatant being much greater than that of the microsomes.