scholarly journals Cyanogen bromide cleavage of proteins in sodium dodecyl sulphate/polyacrylamide gels Diagonal peptide mapping of proteins from epidermis

1981 ◽  
Vol 197 (3) ◽  
pp. 591-597 ◽  
Author(s):  
J D Lonsdale-Eccles ◽  
A M Lynley ◽  
B A Dale
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Distinct Group ◽  
Newborn Mouse ◽  
Polyacrylamide Gels ◽  
Structural Protein ◽  
Human Proteins ◽  
Methionine Residues ◽  
The Individual

A two-dimensional electrophoretic procedure employing CNBr has been devised for the analysis of proteins in sodium dodecyl sulphate/polyacrylamide gels. The technique allows the detection of an unusual class of epidermal proteins that lack methionine. The proteins have been identified by this method in newborn mouse, rat, and rabbit, because they are stable in the presence of CNBr and consequently lie on a diagonal. Adult human epidermis also contains CNBr-stable proteins, but in lesser amounts than in the newborn rabbit or newborn rodents. The methionine-containing proteins (i.e., the keratins) are degraded by CNBr into a series of unique and characteristics peptides which lie below the diagonal. Inter- and intra-species similarities and differences exist between the individual keratins, depending on the number and distribution of their methionine residues. The peptide-map patterns for the rodent and lagomorph proteins are more similar to each other than to that for the human proteins. The maps for rat and rabbit skin proteins are the most similar. We conclude that the epidermal keratins are a closely related, yet individually distinct, group of proteins that are found in conjunction with a class of proteins that lack methionine. The latter proteins are related to the histidine-rich basic protein, an epidermal structural protein that aggregates with keratin filaments.

scholarly journals The molybdenum–iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ‘mapping’

1976 ◽  
Vol 155 (2) ◽  
pp. 383-389 ◽  
Author(s):  
C Kennedy ◽  
R R. Eady ◽  
E Kondorosi ◽  
D K Rekosh
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Sodium Dodecyl Sulphate ◽  
Azotobacter Vinelandii ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Rhizobium Japonicum ◽  
Fe Protein

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide ‘maps’ after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.

scholarly journals Properties of pig synovial collagenase

1980 ◽  
Vol 189 (2) ◽  
pp. 349-357 ◽  
Author(s):  
J A Tyler ◽  
T E Cawston
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Type Ii ◽  
Polyacrylamide Gels ◽  
Optimum Ph ◽  
Detectable Difference

1. Properties of a purified chemically activated form of pig synovial collagenase were examined and compared with a spontaneously active form of the enzyme. 2. The active enzyme has a specific activity of 53 000 units (microgram/min)/mg, a mol.wt. of 44 000 (by sodium dodecyl sulphate/polyarcylamide-gel electrophoresis in 2-mercaptoethanol) and pI 5.2 (by isoelectric focusing in polyacrylamide gels). 3. The activity has the characteristics of a metalloproteinase that degrades types I and III soluble or insoluble collagens in preference to type II, at an optimum pH of 6.5-8.5. 4. There is no detectable difference in these properties between the chemically activated and spontaneously active form of collagenase.

scholarly journals The action of progesterone and diethylstilboestrol on the dehydrogenase and esterase activities of a purified aldehyde dehydrogenase from rabbit liver

1977 ◽  
Vol 161 (1) ◽  
pp. 123-130 ◽  
Author(s):  
R J S Julian
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Rabbit Liver ◽  
Polyacrylamide Gels ◽  
Catalytically Active ◽  
Steroid Sensitive ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of ◽  
Esterase Activities

A steroid-sensitive aldehyde dehydrogenase (EC 1.2.1.3) was purified from rabbit liver and is homogeneous by the criterion of electrophoresis in polyacrylamide gels with or without sodium dodecyl sulphate. The enzyme is tetrameric, of subunit mo.wt. 48 300, and contains no tightly bound zinc. The fluorescence of the protein is decreased in the presence of progesterone, which is inhibitory to the reactions catalysed by the enzyme. When NADH is bound to the enzyme, the fluorescence of the coenzyme is augmented to an extent independent of the presence of steroids or acetaldehyde. The purified enzyme catalyses the oxidation of acetaldehyde and glucuronolactone, and the hydrolysis of 4-nitrophenyl acetate. Each of these reactions is inhibited by progesterone in such a manner as to suggest the formation of a catalytically active enzyme-hormone complex. Diethylstilboestrol inhibits the hydrolysis of esters by this enzyme, but stimulates the oxidation of aldehydes, except at low aldehyde concentrations; the ligand is then inhibitory. NADH inhibits the hydrolysis of 4-nitrophenyl acetate by the enzyme in a partially competitive fashion.

scholarly journals Extraction methods and test techniques for detection of vegetable proteins in meat products:I. Qualitative detection of soya derivatives

1976 ◽  
Vol 76 (3) ◽  
pp. 329-336 ◽  
Author(s):  
N. ST G. Hyslop
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Vegetable Protein ◽  
Sodium Dodecyl ◽  
Meat Products ◽  
Extraction Methods ◽  
Soya Bean ◽  
Animal Origin ◽  
Polyacrylamide Gels ◽  
Migration Rates ◽  
Qualitative Detection

SUMMARYExtracts of 3 soya bean preparations, used commercially in certain countries to replace part of the meat in popular meat products, were made by treatment with (i) sodium dodecyl sulphate, (ii) Triton-X100 or (iii) n–Butanol. Similar extracts were made from beef and pork.All extracts were examined by electrophoretic and immunological techniques. Stained polyacrylamide gels revealed distinctive protein bands after electrophoresis. The migration rates of corresponding bands differed between beef and pork extracts. However, the migration rates of vegetable bands revealed certain similarities, but differed very greatly from those of animal origin. Characteristic fast-migrating S-bands were distinguishable only in extracts of vegetable protein. Immunodiffusion tests, using antisera produced in rabbits against each extract, revealed varying degrees of similarity between extracts of vegetable origin, but the antisera were specific for either vegetable or animal protein.

scholarly journals The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

1972 ◽  
Vol 126 (3) ◽  
pp. 553-560 ◽  
Author(s):  
I. P. Griffith
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Polyacrylamide Gels ◽  
Gel Method ◽  
Molecular Weights ◽  
Before And After ◽  
Cross Links ◽  
Polyacrylamide Gel Method

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate–polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate–polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.

scholarly journals Phosphoenolpyruvate carboxykinase from guinea-pig liver mitochondria. Immunological evidence for increase in enzyme amount during neonatal development

1984 ◽  
Vol 221 (1) ◽  
pp. 105-111 ◽  
Author(s):  
S Wicheanvonagoon ◽  
I J Arinze
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Liver Mitochondria ◽  
Polyacrylamide Gels ◽  
Neonatal Development ◽  
Pig Liver ◽  
Guinea Pig Liver

Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.

scholarly journals Separation at neutral pH value of 32P-labelled proteins in a membrane preparation from ox brain. Partial characterization of a component of the sodium-plus-potassium ion-activated adenosine triphosphatase

1974 ◽  
Vol 137 (2) ◽  
pp. 253-262 ◽  
Author(s):  
Denis R. Alexander ◽  
Richard Rodnight
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Ph Value ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gels ◽  
Ph Profile ◽  
Fast Running ◽  
Neutral Conditions ◽  
Strong Acids

1. Ox brain microsomal fractions were labelled with [32P]ATP in the presence of Na+ and the reaction was stopped with sodium dodecyl sulphate. The Na+-dependent bound phosphate was isolated on Sephadex G-25 and by acetone precipitation. The bound phosphate isolated under these neutral conditions was labile to hydroxylamine and gave the same pH profile of hydrolysis as that isolated by precipitation with strong acids. 2. When membrane protein was labelled with [32P]ATP, solubilized with sodium dodecyl sulphate and fractionated on Sepharose 6B, the Na+-dependent label emerged in a peak corresponding to protein of molecular weight 570000–580000. On fractionation of this protein peak on polyacrylamide gels containing detergent and urea, the Na+-dependent label occurred in a single band corresponding to a protein of molecular weight 102000. 3. Fractionation on Sepharose 6B of protein labelled with [32P]ATP in the absence of Na+ revealed three labelled peaks, one of which corresponded in position to the Na+-dependent label. Electrophoresis of this peak material on polyacrylamide gels showed that most of the label occurred in two fast-running bands. Cyclic AMP stimulated the labelling in these two bands, but had no effect on the labelling of the band corresponding in position to the Na+-dependent label. 4. Di-isopropyl [32P]phosphorofluoridate also labelled the band corresponding to the Na+-dependent label on gel electrophoresis. The labelling of this band by the reagent was inhibited by 50–60% by 3mm-ATP, but there was no evidence to suggest that the group labelled is normally phosphorylated by ATP.

scholarly journals The instantaneous monitoring of polyacrylamide gels during electrophoresis

1976 ◽  
Vol 159 (3) ◽  
pp. 743-748 ◽  
Author(s):  
A Elliott
Keyword(s):  
Refractive Index ◽  
Sodium Dodecyl Sulphate ◽  
Optical Method ◽  
Sodium Dodecyl ◽  
Dark Field ◽  
Polyacrylamide Gels ◽  
Optimal Conditions ◽  
Quantitative Estimates ◽  
Coomassie Blue ◽  
Index Changes

The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel.

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