scholarly journals Enzyme forms produced from aspartate transcarbamoylase by digestion with trypsin

1973 ◽  
Vol 135 (1) ◽  
pp. 125-132 ◽  
Author(s):  
Elizabeth Heyde ◽  
A. Nagabhushanam ◽  
S. Venkataraman
Keyword(s):  
Gel Electrophoresis ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Catalytic Subunit ◽  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Allosteric Effectors ◽  
Activity Measurements ◽  
Polypeptide Chains

1. The time-course of tryptic hydrolysis of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) was followed by activity measurements in the presence and absence of allosteric effectors, and by polyacrylamide-gel electrophoresis. 2. Two proteins with enzyme activity are formed in this way from native enzyme, and the isolation and some properties of these species are reported. The larger protein (10.6S) resembles native enzyme in that it contains regulatory subunits and is sensitive to allosteric effectors, as well as in a more detailed kinetic investigation. It appears from the time-course of tryptic digestion to be an intermediate in the formation of a catalytic subunit (5.5S) which is similar to, but not identical with, the catalytic subunit produced by mercurial treatment of the native enzyme. 3. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of the different enzyme forms demonstrates that trypsin can hydrolyse bonds in the catalytic polypeptide chains as well as completely remove the regulatory polypeptide chains. 4. Both preparations of catalytic subunit can recombine with regulatory subunit to form enzymes which resemble the native enzyme in being activated by ATP, although they do not appear to be inhibited by CTP. 5. This study is consistent with the models of the enzyme that propose that the catalytic subunits are held together in the native enzyme by three pairs of regulatory polypeptide chains.

Urease. X. A study by gel electrophoresis of its dissociation by sodium dodecyl sulfate

1969 ◽  
Vol 47 (10) ◽  
pp. 989-991 ◽  
Author(s):  
D. P. Blattler ◽  
George Gorin
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Dodecyl Sulfate ◽  
Polypeptide Chains

Urease, m.w. 480 000, treated with an excess of sodium dodecyl sulfate is converted to a product of greatly increased mobility in polyacrylamide gel electrophoresis. We estimate its weight to be about 80 000. Treatment with excess thiol and detergent yielded the same product as detergent alone, indicating that the subunit or subunits do not contain polypeptide chains linked by disulfide bonds.

scholarly journals Affinity-chromatographic purification of S-adenosyl-l-homocysteine hydrolase. Some properties of the enzyme from rat liver

1981 ◽  
Vol 193 (2) ◽  
pp. 503-512 ◽  
Author(s):  
E O Kajander ◽  
A M Raina
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Adenosine Kinase ◽  
Native Enzyme ◽  
Apparent Homogeneity ◽  
A Subunit ◽  
Adenosine Derivatives ◽  
Pore Gradient

S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose. The purified enzyme was free from adenosine kinase and adenosine deaminase activities and was homogeneous on SDS/polyacrylamide-gel electrophoresis which gave a subunit mol.wt. of 47 000. The native enzyme showed some microheterogeneity on polyacrylamide-gel electrophoresis under increased-resolution conditions but was homogeneous on isoelectric focusing (pI 5.6). The molecular weight of the native enzyme was about 220 000 as judged by pore-gradient electrophoresis. The native enzyme bound adenosine tightly and showed Km values of 0.6 microM, 0.9 microM and 60 microM for adenosine, S-adenosyl-L-homocysteine and L-homocysteine respectively. The enzyme was rapidly inactivated when incubated in the presence of adenosine, S-adenosyl-L-homocysteine or several adenosine derivatives or analogues. Inactivation took place both at 0 and 37 degrees C. Freezing in the absence of glycerol resulted in the appearance of dissociation products of the oligomeric protein. Multimer formation was observed at low thiol concentrations.

scholarly journals Isolation of a human hepatic 60 kDa aspartylglucosaminidase consisting of three non-identical polypeptides

1989 ◽  
Vol 262 (1) ◽  
pp. 189-194 ◽  
Author(s):  
M Baumann ◽  
L Peltonen ◽  
P Aula ◽  
N Kalkkinen
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Inherited Disease ◽  
Ph Optimum ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Molecular Masses ◽  
Polypeptide Chains

We have characterized the properties of human aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which is deficient in the human inherited disease aspartylglucosaminuria. The purification procedure from human liver included affinity chromatography, gel filtration, strong-anion- and strong-cation-exchange h.p.l.c., chromatofocusing and reverse-phase h.p.l.c. In a denaturing SDS/polyacrylamide-gel electrophoresis, the 6600-fold purified enzyme was shown to be composed of three non-identical inactive polypeptide chains of molecular masses 24, 18 and 17 kDa. In a native polyacrylamide-gel electrophoresis, these polypeptide chains ran as one active enzyme complex. As judged from the elution position of the native enzyme in a Biogel P-100 gel filtration, the approximate molecular mass of this complex was 60 kDa. The enzyme had a pI of 5.7, a pH optimum at 6, of 0.48 mM and a specific activity of 200,000 nkat for the substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose. The enzyme showed a 57% loss of activity at 60 degrees C after 45 h but was practically inactive after incubation at 72 degrees C for a few minutes. The molecular structure, Km and specific activity as well as the thermostability of the enzyme described here are different from those reported previously for human aspartylglucosaminidase.

Proteinases secreted by Fasciola hepatica: time course of the inhibitory effect of serum from experimentally infected rabbits demonstrated by gelatin-substrate polyacrylamide gel electrophoresis

1997 ◽  
Vol 71 (4) ◽  
pp. 333-338 ◽  
Author(s):  
L. Piacenza ◽  
D. Acosta ◽  
A. Dowd ◽  
S. McGonicle ◽  
J. Dalton ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Neutralizing Antibodies ◽  
Time Course ◽  
Fasciola Hepatica ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Group 4 ◽  
Group 3 ◽  
Group 2 ◽  
Group 1

AbstractFasciola hepatica secretes proteolytic enzymes to aid it to penetrate and migrate through the host tissues. Two of these proteinases have been purified and shown to be cathepsin L-like, and are termed, CL1 (27.5 kD) and CL2 (29 kD). The immunogenicity of these proteinases was investigated over the course of an experimental infection and following drug treatment. Four groups of rabbits were studied: group 1: orally infected with 50 metacercariae; group 2: infected and treated 8 weeks after infection; group 3: infected, treated at week 8 and reinfected at week 13 and group 4: non-infected control group. Sera were collected weekly from each group until week 20 postinfection. CL1 and CL2 were incubated with the different sera and then analysed by gelatin substrate polyacrylamide gel electrophoresis (GS-PAGE). Analysis of groups 1, 2 and 3 showed that CL1 and CL2 neutralizing antibodies appear at week 5 post-infection. In group 1, these remained throughout the 20 weeks of infection. In group 2, neutralizing antibodies disappeared at week 13, that is, 5 weeks after anti-Fasciola treatment. In group 3, CL1 and CL2 neutralizing antibodies disappeared at week 13 but reappeared by week 15, that is 2 weeks after reinfection. Pooled sera from group 4, showed no inhibitory capacity. ELISA results using CL1 and CL2 as antigen, correlate very well with the inhibitiory time course observed by GS-PAGE. These results suggest that purified cathepsin Ls are antigenic molecules recognized early in the infective process and capable of inducing a specific humoral response, strong enough to neutralize, at least partially, their enzymatic activity.

scholarly journals Quaternary structure of erythrocruorin from the nematode Ascaris suum. Evidence for unsaturated haem-binding sites

1987 ◽  
Vol 242 (3) ◽  
pp. 689-694 ◽  
Author(s):  
S Darawshe ◽  
Y Tsafadyah ◽  
E Daniel
Keyword(s):  
Gel Electrophoresis ◽  
Protein Concentration ◽  
Quaternary Structure ◽  
Binding Capacity ◽  
Ascaris Suum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Polypeptide Chains

The quaternary structure of erythrocruorin from the nematode Ascaris suum was studied. The native protein had a sedimentation coefficient, at a protein concentration of 1 mg/ml, of 11.6 +/- 0.3 S and an Mr, as determined by sedimentation equilibrium, of 332,000 +/- 17,000. SDS/polyacrylamide-gel electrophoresis gave one band with a mobility corresponding to an Mr of 43,000 +/- 2000. The Mr of the polypeptide chain was determined to be 41,600 +/- 1,500 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. Cross-linking with glutaraldehyde followed by SDS/polyacrylamide-gel electrophoresis yielded a maximal number of eight bands. The haem content of Ascaris erythrocruorin was observed to vary from one preparation to another. This finding was shown to be due to non-realization of the full binding capacity for haem. By titration with haemin, the haem content was found to attain a maximal value of 2.86 +/- 0.14%, corresponding to a minimal Mr per haem group of 21,000 +/- 1,000. Our findings indicate that Ascaris suum erythrocruorin is composed of eight identical polypeptide chains, carrying two haem sites each.

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 Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution.

1977 ◽  
Vol 146 (1) ◽  
pp. 257-270 ◽  
Author(s):  
M K Pangburn ◽  
R D Schreiber ◽  
H J Müller-Eberhard
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Normal Serum ◽  
Polyacrylamide Gel ◽  
Human Complement ◽  
Serum Enzyme ◽  
Absolute Requirement ◽  
Polypeptide Chains

The complement regulatory enzyme, C3b inactivator (C3bINA), has been purified from human serum by affinity chromatography on an anti-C3bINA Sepharose column. Subsequent chromatography on DEAE-cellulose and removal of IgG with anti-IgG Sepharose resulted in a product which was found to be homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecule is composed of two disulfide bonded polypeptide chains with mol wt of 50,000 and 38,000 daltons. Human CobINA was found to be a glycoprotein containing at least 10.7% carbohydrate and to have a normal serum concentration of 34 +/- 7 mug/ml (mean +/- 1 SD). Highly purified C3bINA cleaved neither free C3b nor free C4b if trace amounts of contaminating beta1H were removed from these proteins with anti-beta1H Sepharose. However, in the presence of highly purified beta1H and C3bINA, both C3bIna, both C3b and C4b were cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their structure. The action of C3bINA and beta1H on C3b produced two fragments of the alpha1-chain which did not dissociate without reduction of the molecule. These fragments have mol wt of 67,000 and 40,000 daltons. The action of C3bINA and beta1H on C4b resulted in cleavage of the alpha'-chain giving rise to the 150,000-dalton C4c and the 49,000-dalton C4d fragments which dissociated without reduction. To produce from C3b the immunochemically defined C3c and C3d, fragments, the action of an additional serum enzyme appears to be required, the effect of which can be mimicked by trypsin.

Ultrastructural localization of specific nucleoprotein fractions

1978 ◽  
Vol 36 (2) ◽  
pp. 204-205
Author(s):  
G. L. Brown
Keyword(s):  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultrastructural Localization ◽  
Polyacrylamide Gel ◽  
Acid Extraction ◽  
Acid Solutions ◽  
Low Salt ◽  
Liver Nuclei ◽  
Phosphate Groups

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

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