scholarly journals An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88° C

1987 ◽  
Vol 247 (1) ◽  
pp. 121-133 ◽  
Author(s):  
D A Cowan ◽  
K A Smolenski ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Permeation Chromatography ◽  
Polyacrylamide Gel ◽  
British Library ◽  
Extracellular Proteinase ◽  
Hydrophobic Residues ◽  
The Stability

An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5.

scholarly journals The quaternary structure of wheat-germ aspartate transcarbamoylase

1982 ◽  
Vol 203 (2) ◽  
pp. 413-417 ◽  
Author(s):  
R J Yon ◽  
J E Grayson ◽  
A Chawda ◽  
P J Butterworth
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Wheat Germ ◽  
Quaternary Structure ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Aspartate Transcarbamoylase ◽  
Molecular Masses

1. The molecular mass of aspartate transcarbamoylase purified from wheat germ was found to be 101kDa by sucrose-density-gradient centrifugation, 103kDa by gel-filtration chromatography and 108kDa by polyacrylamide-gel electrophoresis. A mean value of 104 +/- 11kDa was obtained by pooling several replicate results from each method. 2. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated a single size of polypeptide chain of mean molecular mass 37 +/- 4kDa. The ratio of the mean molecular masses of the active and denatured enzymes is 2.8.3. When the active enzyme was covalently cross-linked at a low protein concentration by dimethyl suberimidate, and then examined electrophoretically under denaturing conditions, three size species were observed to predominate, of apparent molecular masses 36, 77 and 106kDa respectively. 4. These results indicate that the intact, fully regulatory enzyme is a simple trimer, slightly larger than the trimeric ‘catalytic subunit’ of the aspartate transcarbamoylase from Escherichia coli [Weber (1968) Nature (London) 218, 1116-1118]. The prevalence of trimeric structures amongst carbamoyl-transferase enzymes is discussed.

scholarly journals Polymorphism of Gli-D1 alleles of Kragujevac’s winter wheat cultivars (Triticum aestivum L.)

Genetika ◽  
2007 ◽  
Vol 39 (2) ◽  
pp. 273-282
Author(s):  
Desimir Knezevic ◽  
Aleksandra Novoselskaya-Dragovich
Keyword(s):  
Triticum Aestivum ◽  
Winter Wheat ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Triticum Aestivum L ◽  
Polyacrylamide Gel ◽  
Wheat Cultivars ◽  
Number Of Components ◽  
Winter Wheat Cultivars

Composition of gliadins encoded by Gli-D1 allele as well polymorphisms of Gli-D1 allele investigated in 25 wheat cultivars by using acid polyacrylamide gel electrophoresis. Electrophoregrams obtained by polyacrylamide gel electrophoresis were used for estimation variability of gliadin components and identification of gliadin blocks. Five gliadin blocks encoded by different alleles at Gli-D1 locus were apparently expressed and identified. Gliadin blocks differed according to number of components and their molecular mass. Variability of determined block components indicates that existing polymorphisms of gliadins alleles. Frequency of identified 5 alleles at Gli-D1 locus was in ratio from 4% to 52%. The highest frequency of b allele and the of g allele was found.

Isozymes as markers for identification of tissue culture and greenhouse-grown strawberry cultivars

1991 ◽  
Vol 71 (4) ◽  
pp. 1195-1201 ◽  
Author(s):  
N. S. Nehra ◽  
K. K. Kartha ◽  
C. Stushnoff
Keyword(s):  
Tissue Culture ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Meristem Culture ◽  
Banding Patterns ◽  
Extraction Buffer ◽  
Leaf Tissues ◽  
The Stability ◽  
Strawberry Cultivars

Polyacrylamide gel electrophoresis (PAGE) was used for analysis of isozyme banding patterns of leucine aminopeptidase (LAP), phosphoglucomutase (PGM), phosphoglucoisomerase (PGI), esterase (EST) and 6-phosphogluconate dehydrogenase (6-PGD) in strawberry leaves. The extracts prepared from young leaf tissues using polytron homogenization and an extraction buffer containing 15 mg ml−1 dithiothreitol (DTT) and 10% insoluble polyvinylpyrrolidone (PVP-6755) gave best resolution for these enzymes. The influence of plant age and various growing environments on the stability of isozyme phenotypes was examined. The isozyme banding patterns of 6-PGD were found to vary with the change in growing environment as well as age of the plants. EST produced different banding patterns in greenhouse and tissue culture leaves. However, the isozyme phenotypes of LAP, PGM and PGI remained stable under all the conditions tested. Using a combination of these three stable enzymes, it was possible to distinguish eight strawberry cultivars under both tissue culture and greenhouse conditions. Key words: Fragaria × ananassa Duch., meristem culture, polyacrylamide gel electrophoresis

scholarly journals The small dermatan sulphate proteoglycans synthesized by fibroblasts derived from skin, synovium and gingiva show tissue-related heterogeneity

1988 ◽  
Vol 256 (1) ◽  
pp. 35-40 ◽  
Author(s):  
H Larjava ◽  
J Heino ◽  
T Krusius ◽  
E Vuorio ◽  
M Tammi
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gingival Fibroblasts ◽  
Dermatan Sulphate ◽  
Different Tissues

Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix.

scholarly journals Purification of the constituent enzyme fractions of mycobacillin synthetase

1986 ◽  
Vol 235 (1) ◽  
pp. 81-85 ◽  
Author(s):  
S K Ghosh ◽  
N K Mukhopadhyay ◽  
S Majumder ◽  
S K Bose
Keyword(s):  
Gel Electrophoresis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Sucrose Density Gradient Centrifugation ◽  
Final Purification

The final purification of the three-fraction enzyme complex mycobacillin synthetase was done by hydroxyapatite column chromatography and sucrose-density-gradient centrifugation; each of the fractions obtained migrates as a single component in SDS/polyacrylamide-gel electrophoresis and gel electrofocusing. The Mr of the enzyme fractions A, B and C by gel filtration is 260 000, 190 000 and 105 000, and that by SDS/polyacrylamide-gel electrophoresis is 252 000, 198 000 and 108 000 respectively. None of the enzyme fractions appears to possess subunit structure.

scholarly journals Presence of a high-molecular-weight form of catalase in enzyme purified from mouse liver

1977 ◽  
Vol 163 (3) ◽  
pp. 449-453 ◽  
Author(s):  
M B Baird ◽  
H R Massie ◽  
L S Birnbaum
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
High Molecular Weight Form ◽  
High Molecular Weight Material ◽  
Molecular Weight Form

Ultracentrifugation studies of purified mouse hepatic catalase revealed that 5-7% of the total material consists of a form with a higher molecular weight than the bulk of the catalase. The two components were separated by sucrose-gradient centrifugation. Polyacrylamide-gel electrophoresis (in borate buffer) demonstrated that high-molecular-weight catalase is enriched in a more slowly migrating component, and sodium dodecyl sulphate/polyacrylamide gel-electrophoresis demonstrated that the molecular weight of the subunits of the high-molecular-weight material is identical with that of the subunits of the major form. These results suggest that high-molecular-weight catalase consists of subunits that are not markedly distinct from those present in the normal catalase tetramer.

Does the B820 Subcomplex of the B880 Complex Retain Carotenoids?

1996 ◽  
Vol 51 (5-6) ◽  
pp. 309-318 ◽  
Author(s):  
Andrey Moskalenko ◽  
Olga Toropygina ◽  
Nina Kuznetsova
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Rhodospirillum Rubrum ◽  
Complex Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
The Common

Abstract A study is reported on the modification of the B880-RC assembly of Chromatium minutissimum during octyl-β-D -glucopyranoside/dodecyl-β-D -maltoside/Deriphat polyacrylamide gel electrophoresis followed by electroelution with dodecyl-β-D-maltoside and high performance liquid chromatography with octyl-β-D-glucopyranoside according to the method developed by Kerfeld et al. (Biochim. Biophys. Acta 1185, 193-202 [1994a]) for isolation of the B820 subcomplexes of Chromatium purpuratum. The B880-RC assembly of Chromatium minutissimum isolated by electrophoresis was contaminated by the B 800-850 complex. It was further separated into four components, three of which were in agreement with the cited work: (i) colorless contaminations, (ii) the B880-RC assembly, (iii) the B 800-850 complex. In contrast with Kerfeld et al. (1994a), the fourth band was a band of free pigments (Bchl or Bchl-t-carotenoids) which had the same molecular mass as the B820 subcomplex of Chromatium purpuratum. For comparison, the B880-RC enriched fraction of Rhodospirillum rubrum modified by lyophilization in the presence of octyl-β-D-glucopyranoside with or w ithout carotenoids was separated by high performance liquid chromatography with octyl-β-D-glucopyranoside. The apparent molecular mass of the B820 subcomplex was 30 kD a for the sample without carotenoids and 245 kD a for that with carotenoids. The common principles of organization of the B880 complex, the interaction of the B 800- 850 complex with the B880-RC assembly, the participation of carotenoids in the stabilization of the B880 complex structure and the ability of different isolation steps to modify the structure of the B880 complex are discussed. It was concluded that there are other explanations for the presence of carotenoids in the B820 subcomplex. Hence, the question of whether the B820 subcomplex retains carotenoids remains open.

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