A high resolution PAS stain for polyacrylamide gel electrophoresis

Kapitany, R. A.; Zebrowski, E.J.

doi:10.1016/0003-2697(73)90202-9

Cited by 397 publications

(130 citation statements)

References 12 publications

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“…Polyacrylamide slab gel electrophoresis was done according to Laemmli (17). Proteins were stained with Coomassie blue R250 or G250 and carbohydrates were stained with Schiff reagent (18).…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase i).

Levin¹,

Skidgel²,

Erdös³

1982

Proc. Natl. Acad. Sci. U.S.A.

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Carboxypeptidase N (kininase I, arginine carboxypeptidase; EC 3.4.17.3) cleaves COOH-terminal basic amino acids of kinins, anaphylatoxins, and other peptides. The tetrameric enzyme of Mr 280,000 was purified from.human plasma by ion-exchange and arginine-Sepharose affinity chromatography. Treatment with 3 M guanidine dissociated the enzyme into subunits of 83,000 and 48,000 molecular weight, which were separated and purified by gel filtration or affinity chromatography. When tested with hippurylarginine, hippurylargininic acid, benzoylalanyllysine, or bradyldkini the Mr 48,000 subunit was as active as the intact enzyme-and was easily distinguished from human pancreatic carboxypeptidase B (EC 3.4.17.2). However, the Mr 48,000 subunit was less stable at acid pH or at 370C than the intact enzyme was. The carbohydrate-containing Mr 83;000 subunit was enzymatically inactive but stabilized the Mr 48,000 subunit at 37°C. Trypsin, plasmin, and plasma or urinary kallikrein cleaved carboxypeptidase N into lower molecular weight active fragments, which were unstable at 37°C. Cleavage of the Mr 48,000 subunit with the same enzymes increased activity and yielded fragments OfMr 29,000 or less. Antibodies to the Mr 83,000 or Mr 48,000 subunits crossreacted with the intact enzyme, and antibody to carboxypeptidase N also recognized both subunits. However, antibody' to the Mr 83,000 subunit did' not recognize the Mr 48,000 subunit and antibody to the Mr 48,000 subunit did not crossreact with the Mr 83,000 subunit. Thus, this study indicates that carboxypeptidase N is composed of two immunologically distinct subunits, a Mr 48,000 subunit that is responsible for the. enzymatic activity and a-Mr 83,000 subunit that may stabilize the enzyme in blood.Carboxypeptidase N or arginine carboxypeptidase (EC 3.4.17.3) is an important inactivator ofpotent peptides such as kinins (1), anaphylatoxins (2, 3), and fibrinopeptides (1). It also cleaves COOH-terminal basic amino acids of a variety of other peptide substrates (1, 4). The enzyme exists in plasma as a Mr 280,000 tetrameric complex, even when plasma is stored for several months (5, 6). Purification of the enzyme from plasma on ionexchange and affinity columns leads to an unstable preparation (5-7); this can be stabilized with protease inhibitors (7). Thus, dilution of naturally occurring inhibitors or simultaneous adsorption ofthe enzyme and contaminating proteases (e.g., plasmin) during purification leads to the appearance of lower molecular weight derivatives (5-8).The aims of the. present study were (i) to dissociate purified carboxypeptidase N and isolate its subunits; (ii) to determine the enzymatic activity, stability, and function of the isolated subunits; (iii) to determine the effect of various proteolytic enzymes on.the activity and stability ofboth the intact enzyme and its. subunits. (13). Enzyme Activity. The activity of carboxypeptidase N was measured in a spectrophotometer with the ester, hippurylargininic acid, or the peptide, Bz-Ala-Lys, substrate at 254 nm ...

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“…Polyacrylamide slab gel electrophoresis was done according to Laemmli (17). Proteins were stained with Coomassie blue R250 or G250 and carbohydrates were stained with Schiff reagent (18).…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase i).

Levin¹,

Skidgel²,

Erdös³

1982

Proc. Natl. Acad. Sci. U.S.A.

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“…Gels were silverstained for protein using a Bio-Rad kit according to the manufacturer's instructions. Staining for carbohydrate was with periodic acid/Schiff s base [23].…”

Section: Sds-pagementioning

confidence: 99%

Plasminogen activator inhibitor from human endothelial cells. Purification and partial characterization

et al. 1987

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An inhibitor of plasminogen activator was purified to apparent homogeneity from human umbilical vein endothelial cell conditioned medium. The purification was achieved by a speedy and simple two-step procedure, without the use of denaturants. The purified protein was a single-chain glycoprotein with apparent molecular mass of 48 kDa.The purified inhibitor had a specific activity of 8500 U/mg protein and the activity could be stimulated about fourteenfold by treatment with denaturants.An antiserum to the purified inhibitor was raised in rabbits. It recognised plasminogen activator inhibitor from platelets and plasma as well as from cultured endothelial cells. The immunoglobulin fraction of the antiserum neutralised the functional activity of the inhibitor from all these sources.The activity of the fibrinolytic system depends on the cleavage of the zymogen plasminogen to form plasmin by plasminogen activators. Two major immunologically distinct types of plasminogen activator, tissue-type (tPA) and urokinase (u-PA) have been identified (reviewed in [l]). t-PA is present in normal resting plasma at a molecular mass of about 110 kDa [2 -41, in contrast to the 65-kDa form isolated from tissues [5]. The high-molecular-mass t-PA in plasma is now known to be an equimolar complex of t-PA with a plasminogen activator inhibitor (PAI) [3, 41. Inhibitors active against both t-PA and u-PA have been discovered in bovine [6] and human [7 -91 endothelial cells, in platelets [lo, 111 and in plasma [3, 12 -141. Immunological cross-reactivity between the bovine endothelial and human platelet inhibitor has been established [15] and this type of PA1 is now designated PAI-1 [16].The bovine endothelial cell PA1 is in an inactive or latent form that is converted to an active form in the presence of denaturants such as SDS and guanidinium hydrochloride [17]. This inhibitor has been purified using SDS/polyacrylamide gel electrophoresis (SDS-PAGE) as the final preparative step; thus the purified product was active [18].In the study reported here, we aimed to purify PA1 from human endothelial cells in the absence of denaturants. T h s Correspondence to

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“…Water-insoluble polysaccharides were directly located as white bands, and water-soluble ones were stained by the periodic acid-Schiff base (PAS) method [13].…”

Section: Isoelectric Focusingmentioning

confidence: 99%

Three kinds of extracellular glucosyltransferases from Streptococcus mutans 6715 (serotype g)

1983

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In addition to the 1,3*-D-glucan synthetase (p1 4.9) and the highly-branched 1,6_cu-D-glucan synthetase (PI 3.9-4.1), Streptococcus mutans 6715 (serotype g) was found to secrete the third glucosyltransferase in multiple forms (PI 5.5-7.0), which exhibited 87% 1,6_cu-bond-, 6% 1,3_cu-bond-and 7% 1,3,6-branchforming activities. The production of this enzyme was extremely enhanced when the organism was grown in Tween IO-supplemented medium. The 3 glucosyltransferases from the same organism were enzymatically and immunologically distinct from each other, and they were commonly found among the serotype g strains.Streptococcus mutans Glucosyltransferase Glucan Tween 80

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A high resolution PAS stain for polyacrylamide gel electrophoresis

Cited by 397 publications

References 12 publications

Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase i).

Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase i).

Plasminogen activator inhibitor from human endothelial cells. Purification and partial characterization

Three kinds of extracellular glucosyltransferases from Streptococcus mutans 6715 (serotype g)

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