scholarly journals Protein aggregation. Studies of larger aggregates of C-phycocyanin

1968 ◽  
Vol 110 (3) ◽  
pp. 457-464 ◽  
Author(s):  
J. J. Lee ◽  
D. S. Berns
Keyword(s):  
Density Gradient ◽  
Size Range ◽  
Gel Filtration ◽  
Fluorescence Emission ◽  
Sucrose Density Gradient ◽  
Denatured Protein ◽  
Elution Pattern ◽  
Green Algal ◽  
Fluorescence Efficiency ◽  
Algal Extracts

Aggregates of phycocyanin sedimenting at 17s, 22s and 27s are demonstrated to constitute more than 40% of crude blue–green-algal extracts, pH6·0 and I0·1, and are retained in highly purified preparations. Sedimentation-velocity studies of the large aggregates as a function of pH are reported. Sucrose-density-gradient experiments performed as a function of time of sedimentation indicate that: (1) with increasing time of sedimentation, the largest aggregates are dissipated at the leading protein boundary and the several phycocyanin species present are not completely resolved; (2) phycocyanin fractions with the largest aggregates exhibit the highest E620/E280 ratio and the largest relative fluorescence efficiency. Gel-filtration experiments with Sephadex G-200 do not resolve the species completely, and reapplication of phycocyanin gel-filtration fractions to the column results in an elution pattern similar to the original, except that there is an enhancement of the allophycocyanin fraction and the amount of denatured protein. Increasing the sedimentation times in a sucrose density gradient also enhances the allophycocyanin fraction. Fluorescence results demonstrate that there are possibly three excitation maxima, one corresponding to the monomer (approx. 600mμ), one for higher aggregates (625–630mμ) and one for the allophycocyanin fraction (approx. 650mμ). Only a single fluorescence-emission band is detected, which is fairly symmetrical and which has a red shift with higher aggregation and with the appearance of allophycocyanin. The appearance of allophycocyanin may be correlated with the irreversible disaggregation of the largest phycocyanin species. It is suggested that the largest protein aggregates are in the size range of the biliprotein aggregates reported in electron microscopy of algal cells.

scholarly journals Protein aggregation. The effect of deuterium oxide on large protein aggregates of C-phycocyanin

1968 ◽  
Vol 110 (3) ◽  
pp. 465-470 ◽  
Author(s):  
J. J. Lee ◽  
D. S. Berns
Keyword(s):  
Density Gradient ◽  
Deuterium Oxide ◽  
Fluorescence Emission ◽  
Sucrose Density Gradient ◽  
Structural Protein ◽  
Visible Absorption ◽  
Large Size ◽  
Fluorescence Efficiency ◽  
Emission Studies

The amount of 11s aggregate in phycocyanin, normally stimulated by hydrophobic forces, is dramatically increased by the presence of deuterium oxide. Proteins in which hydrophobic forces are not proposed as a mechanism for aggregation are unaffected by deuterium oxide. These observations are consistent with the lower critical micelle concentration reported for ionic detergents in deuterium oxide. Phycocyanin samples containing a majority of material sedimenting faster than 11s were also investigated in the presence of deuterium oxide with the following findings: the most rapidly sedimenting species in water buffer is 24s; in deuterium oxide more than 10% of the protein sediments at 67s and substantial amounts of other species with sedimentation coefficients larger than 24s are present. These large quantities of species sedimenting faster than 24s are found in deuterium oxide buffers from pD5·5 to 7·0. Sucrose-density-gradient studies in deuterium oxide at pD6·0 confirm the presence of large amounts of more rapidly sedimenting species. Spectrophotometric studies on fractions from the sucrose-density-gradient experiments indicate with the presence of higher aggregates a red shift of the visible-absorption maximum and an enhancement of the E620/E280 ratio. Fluorescence-emission studies show a greater relative fluorescence efficiency for these higher aggregates and are consistent with the suggested enhancement of higher aggregates in deuterium oxide. The existence of phycocyanin aggregates of such a large size is suggested to be of importance in vivo, with phycocyanin playing a role as a structural protein.

scholarly journals Crystallization and characterization of human liver kynurenine–glyoxylate aminotransferase. Identity with alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase

1980 ◽  
Vol 189 (3) ◽  
pp. 581-590 ◽  
Author(s):  
Etsuo Okuno ◽  
Yohsuke Minatogawa ◽  
Masayuki Nakamura ◽  
Naoki Kamoda ◽  
Junko Nakanishi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Density Gradient ◽  
Human Liver ◽  
Analytical Ultracentrifugation ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Glyoxylate Aminotransferase

Kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s20,w value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine–glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase, serine–pyruvate aminotransferase and alanine–hydroxypyruvate aminotransferase reactions of the enzyme are presented.

scholarly journals Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 155 (1) ◽  
pp. 107-115 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Minatogawa ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Aminotransferase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 799-808
Author(s):  
E.K. Shibuya ◽  
Y. Masui
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Molecular Characteristics ◽  
Small Peptides ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytostatic Factor

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20–30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 × 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.

scholarly journals Organ distribution of rat histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 157 (3) ◽  
pp. 635-641 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Isoelectric Focusing ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Organ Distribution ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Amino Donor

The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.

scholarly journals Reconstitution of an Allophycocyanin Trimer Complex Containing the C-Terminal 21-23 kDa Domain of the Core-Membrane Linker Polypeptide Lcm

1994 ◽  
Vol 49 (5-6) ◽  
pp. 331-336 ◽  
Author(s):  
Lothar Gottschalk ◽  
Friedrich Lottspeich ◽  
Hugo Scheer
Keyword(s):  
Density Gradient ◽  
Fluorescence Emission ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Preparative Scale ◽  
Small Core ◽  
The Core ◽  
Spectral Changes ◽  
Sds Page ◽  
Molecular Masses

Abstract Allophycocyanin (AP) was isolated from extracts of the cyanobacterium Mastigocladus laminosus. A fraction enriched in AP-associated polypeptides with apparent molecular masses of 21 -23 kD a in SDS-PAGE, was isolated on a preparative scale and identified as a homolo­gous mixture of C-terminal fragments of the core-membrane linker polypeptide Lcm. The complex (αAPβAP)3 ·21 -23 kD a was reconstituted and characterized by sucrose density gradient ultracentrifugation, absorption, fluorescence emission and circular dichroism spectroscopy. The 21 -23 kD a polypeptides were found to induce spectral changes in AP similar to those induced by the small core linker polypeptide Lc8.9. Possible functions of the complex in phycobilisomes are discussed.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

Characterization and Partial Purification of Heterodisperse Polysomal RNAs in Normal and Neoplastic Cells

1972 ◽  
Vol 58 (2) ◽  
pp. 71-94
Author(s):  
Ada Sacchi ◽  
Gianni Chinali ◽  
Susetta Pons ◽  
Michela Galdieri ◽  
Piero Cammarano
Keyword(s):  
Size Distribution ◽  
Density Gradient ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Partial Purification ◽  
Messenger Rnas ◽  
Alkaline Ph ◽  
Sedimentation Profile ◽  
Molecular Weights ◽  
Phenol Extraction

The size distribution of cytoplasmic messenger RNAs (m-RNA) has been studied in rat liver and in monodifferentiated cells (mouse reticulocytes and myelomas). It has been found that the RNA which exhibits a « rapid turnover » and a polydisperse profile of radioactivity is refractory to phenol extraction. This property has been exploited to selectively isolate m–RNA from the phenol residue by means of an extraction at an alkaline pH. The sucrose density gradient profiles of m–RNA isolated from monodifferentiated cells show monodisperse peaks having the sedimentation coefficients expected on the basis of the molecular weights of monocistronic messages for α and β chains of hemoglobin (reticulocytes) and L and H chains of immunoglobulin (myelomas). The sedimentation profile of cytoplasmic m–RNA associated with rat liver polysomes shows a much broader distribution, with sedimentation coefficients ranging from 8 S to 28 S.

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