scholarly journals Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G

1976 ◽  
Vol 155 (2) ◽  
pp. 255-263 ◽  
Author(s):  
P M. Starkey ◽  
A J. Barrett
Keyword(s):  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Cathepsin G ◽  
Gel Chromatography ◽  
Human Spleen ◽  
Molecular Weights ◽  
Ph Values ◽  
Brij 35 ◽  
Insoluble Material

1. Human spleen was found to contain proteinases active against azo-casein at neutral and alkaline pH values. 2. The activity was stimulated by high ionic strength and some detergents. 3. Optimal extraction of the proteinases from the tissue was achieved with 1.0M-NaCl containing 0.1% Brij 35 and 0.1% trisodium EDTA. 4. The proteinases were efficiently adsorbed to insoluble material in the absence of salt in the initial stages of purification. 5. Two distinct proteinases were separated by chromatography on DEAE-cellulose, an elastase and a chymotrypsin-like enzyme designated cathepsin G. 6. Both enzymes were highly purified by further column chromatography. 7. The molecular weights of the enzymes were estimated by gel chromatography and sodium dodecyl sulphate-gel electrophoresis. 8. It was shown by isoelectric focusing and gel electrophoresis that both enzymes are cationic proteins that occur in multiple forms.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

scholarly journals α-Crystallin. Fractionation of subunits and sequence studies on an isolated polypeptide

1971 ◽  
Vol 124 (2) ◽  
pp. 345-355 ◽  
Author(s):  
Robert C. Augusteyn ◽  
Abraham Spector
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Cyanogen Bromide ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Terminal Sequence ◽  
Amino Acid Compositions ◽  
Molecular Weights

α-Crystallin was carboxymethylated with radioactive iodoacetic acid in the presence of 7.6m-urea and then separated into six major fractions by chromatography on DEAE-cellulose in 7m-urea. Based on the amino acid compositions, specific radioactivities and sodium dodecyl sulphate–gel electrophoresis of the fractions, it was concluded that α-crystallin contains at least four different subunits: DU1A and DU1B, containing no cysteine; a third component represented by DU2B and DU3 containing one cysteine one cysteine residue per subunit; and DU4, which probably contains two residues of cysteine per subunit. Subunit DU1A was shown to be of sufficient purity for sequence studies. Cyanogen bromide cleavage yielded two peptides, CB-1 and CB-2, in approximately equal amounts as expected. The sum of the molecular weights and amino acid compositions of the peptides were both in excellent agreement with the results obtained for subunit DU1A. The amino acid sequence of the first sixteen residues of peptide CB-1 is: Ser-Leu-Thr-Lys-Asp-Phe-Asp-Glu-Val-Asn-Ile-Asp-Val-Ser-His-Phe-. The sequence of the first seventeen residues of peptide CB-2 is: Asp-Ile-Ala-Ile-Ser-His-Pro-Trp-Ile-Arg-Pro-Ser-Phe-Phe-Glu-Phe-His-. The N-terminal sequence of subunit DU1A was shown to be N-acetylmethionine followed by peptide CB-2.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH

1978 ◽  
Vol 173 (2) ◽  
pp. 553-563 ◽  
Author(s):  
P R Flanagan ◽  
G G Forstner
Keyword(s):  
New Species ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Ph ◽  
Precipitin Line ◽  
Molecular Weights ◽  
Ph Values

Maltase-glucoamylase, a microvillous membrane ectoenzyme, was solubilized from rat intestinal mucosa by digestion with papain and subsequently purified to homogeneity with an overall yield of 10–20%. An antibody to the purified enzyme formed a single precipitin line in immunodiffusion experiments with an intestinal homogenate. The enzyme was shown to be an acidic glycoprotein (20% sugar by weight) which contained low amounts of cysteine and no sialic acid. At pH3–6, maltase activity was slowly lost, but the enzyme was re-activated by re-adjustment of the pH to neutrality. However, in the presence of sodium dodecyl sulphate, acid pH values inactivated maltase irreversibly, and at the same time converted the enzyme (mol.wt. 500000 approx.) into five new species with apparent molecular weights ranging from 134000 to 480000 as judged by polyacrylamide-gel electrophoresis. The same five fragments were also formed by boiling the enzyme for brief periods in the presence of sodium dodecyl sulphate or urea either with or without reducing agents. The dissociated species were stable on re-electrophoresis, and amino acid analysis showed them to be very similar to each other and to the original enzyme. The bands migrated anomalously on polyacrylamide gels of different concentration, thereby preventing the assignment of precise molecular weights. It is possible that the five species may represent stable aggregates of a common monomer of the enzyme.

Structural Studies on Antigen E of Ragweed Pollen

1973 ◽  
Vol 51 (9) ◽  
pp. 1275-1280 ◽  
Author(s):  
B. W. Griffiths
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reducing Agent ◽  
Ragweed Pollen ◽  
Gel Chromatography ◽  
Structural Studies ◽  
Molecular Weights ◽  
Polypeptide Chains

The analysis of antigen E of ragweed pollen by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) resulted in the demonstration of two subunits of molecular weights of about 20 000 and 14 500. These results confirm earlier studies on antigen E by gel chromatography that demonstrated a two-polypeptide subunit composition. Treatment of antigen E with 1% SDS (in the absence of reducing agent) resulted in the dissociation of the two polypeptide chains, which indicates that the bonds bridging the subunits are non-covalent in nature.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

Effect of detergents and chemicals on purified vaccinia virus: analysis by SDS polyacrylamide gel electrophoresis and electron microscopy

1977 ◽  
Vol 23 (3) ◽  
pp. 240-252 ◽  
Author(s):  
J. Boisvert ◽  
T. Yamamoto
Keyword(s):  
Electron Microscopy ◽  
Vaccinia Virus ◽  
Gel Electrophoresis ◽  
Alkali Treatment ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ammonium Bromide ◽  
Molecular Weights ◽  
Viral Particles

Vaccinia virus particles were dissociated into their constituent polypeptides and analysed by sodium dodecyl sulfate (SDS) gel electrophoresis. Thirty-three distinct polypeptide bands were identified and their molecular weights ranged between 11 000 and 150 000 daltons.Specific staining of gels containing polypeptides of dissociated virions revealed the presence of eight glycopeptides. No lipopeptides were detected.Analysis of chemical extracts (urea, guanidine hydrochloride, and alkali treatment) of the virus by SDS gel electrophoresis indicated that a total of 10 to 14 different polypeptides ranging in molecular weights from 11 000 to 70 000 daltons were solubilized.Analysis of detergent extracts and of the remains of extracted viral particles has shown that the detergent Nonidet P-40 (NP-40) solubilized a total of 11 polypeptides of which 6 were glycopeptides. The other detergents sodium deoxycholate (SDC) and cetyl trimethyl ammonium bromide (CTAB) were not as selective, both solubilizing more than 25 of the polypeptides composing the virus. Gel electrophoresis results also indicated that most of the small molecular weight (11 000–70 000 daltons) polypeptides were readily solubilized by NP-40, SDC, and CTAB, while those with molecular weights of 70 000 daltons and higher were not well solubilized.The effects of detergents were also analysed by electron microscopy. Evidence was obtained for subpopulations of viral particles having different susceptibility to detergent extraction.

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

Subunit studies of coupling factor 1 of bean chloroplasts

1976 ◽  
Vol 54 (5) ◽  
pp. 481-487 ◽  
Author(s):  
M. P. Silvanovich ◽  
R. D. Hill
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coupling Factor ◽  
Leaf Extracts ◽  
Molecular Weights ◽  
Major Species ◽  
Chloroplast Coupling Factor ◽  
Coupling Factors

A bean chloroplast coupling factor (CF1) with latent Ca2+-dependent ATPase activity was studied. Immunodiffusion of bean (Phaseolus vulgaris) chloroplast and etioplast coupling factors and spinach coupling factor against antiserum to spinach coupling factor showed partial identity of the bean coupling factor with that of spinach. An immunoelectrophoretic comparison, under dissociating conditions, of bean leaf extracts and spinach extracts containing CF1 subunits (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) gave identical results for both extracts. At least six distinct polypeptide species were found. The major species had molecular weights of 42 000, 59 000 and 63 000 daltons. Amino acid analysis of electrophoretically purified bean CF1 gave results similar to those published for spinach CF1.

Apolipoprotein C-II deficiency associated with nonfunctional mutant forms of apolipoprotein C-II

1984 ◽  
Vol 62 (9) ◽  
pp. 847-852 ◽  
Author(s):  
Graham F. Maguire ◽  
J. Alick Little ◽  
Gary Kakis ◽  
W. Carl Breckenridge
Keyword(s):  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Normal Subjects ◽  
Two Dimensional ◽  
Antibody Technique ◽  
Molecular Weights ◽  
Apolipoprotein C ◽  
Antigen Antibody ◽  
Mutant Forms

Two previously unidentified apolipoproteins (apo) designated apo C-II-X and C-II-Y have been found in plasma of homozygotes and obligate heterozygotes of a family with apo C-II deficiency. Because the plasmas of homozygotes do not activate lipoprotein lipase, apo C-II-X and C-II-Y are apparently nonfunctional. These apolipoproteins have isoelectric focusing points of 5.15 and 5.54, respectively, compared with 4.88 and 4.74 for the known isomorphs, C-II-1 and C-II-2, respectively. They have approximately similar molecular weights to apo C-II-1 and C-II-2 on two-dimensional sodium dodecyl sulphate – glycerol – polyacrylamide slab gel electrophoresis. They do not form insoluble antigen–antibody complexes with antibodies to apo C-II in single antibody immunodiffusion or electroimmunoassay systems. However, using a double-antibody technique in which immunoblotting is coupled with polyacrylamide isoelectric focusing slab gel electrophoresis, apo C-II-1, C-II-2, C-II-X, and C-II-Y have similar reactivity with antibodies to apo C-II. In this family the presence of apo C-II-X and C-II-Y discriminates obligate heterozygotes from normal subjects.

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