A procedure to increase the sensitivity of staining by Coomassie brilliant blue G250-perchloric acid solution

Holbrook, Ian; Leaver, A.G.

doi:10.1016/0003-2697(76)90118-4

Cited by 184 publications

(35 citation statements)

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“…Muramidase-2 activity was assayed by its ability to hydrolyze either of two different substrates, S. faecium peptidoglycan or SDS-washed M. luteus cell walls, both of which are poorly or are not dissolved by muramidase-1 (19 (16). SDS gels containing low amounts of protein were stained with silver by the procedure specified by Bio-Rad Laboratories, Richmond, Calif. Molecular masses were determined on the basis of migration of molecular mass standards (Pharmacia, Inc., Piscataway, N.J.).…”

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The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin

1989

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A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a 0-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a ['4C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.Streptococcus faecium ATCC 9790 (Enterococcius hirae [10]) was previously shown to possess two distinct peptidoglycan hydrolases, which differ from each other in substrate specificity, molecular mass, and, very probably, mechanism of hydrolysis (19). Muramidase-1 was isolated and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose and was shown to occur in a latent 130-kilodalton (kDa) form that can be proteolytically converted to an active 87-kDa form (20, 24). Muramidase-1 was also shown to contain covalently attached monomeric and oligomeric glucose (20) and monomeric 5-mercaptouridine monophosphate (8). In addition, muramidase-1 was shown to processively hydrolyze linear-soluble, un-crosslinked peptidoglycan chains (1).A second peptidoglycan hydrolase (muramidase-2) has been partially purified from culture supernatants by affinity binding to, and elution from, sodium dodecyl sulfate (SDS)-treated cell walls of Micrococcus luteus and has been shown to be a . It was shown to differ from muramidase-1 in substrate specificity, dissolving the cell walls of M. luteus or the acid-insoluble peptidoglycan fraction of S. faecium walls but having little ability to dissolve S. faecium cell walls. Additional data suggest that muramidase-1 and muramidase-2 are separate gene products.We have purified muramidase-2 from the insoluble pellet of disrupted S. faecium cells. Muramidase-2 was extracted with 0.02 N NaOH at 0°C, bound to the acid-insoluble peptidoglycan fraction of S. faecium, and reextracted with alkali. SDS-polyacrylamide gel electrophoresis (PAGE) of fractions highly enriched for muramidase-2 activity showed the presence of two protein bands, at 125 and 75 kDa. Upon elution and renaturation from SDS gels, both protein bands were shown to possess muramidase-2 activity. The two proteins appeared to be biochemically and immunochemically related and may represent different forms ...

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confidence: 99%

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin

1989

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“…The 2nd dimensional analysis is pursued on a 7% SDS-polyacrylamide gel [ 24]. After electrophoresis, the gel was either stained with silver [ 25], or Commassie Blue G [26]. Protein size markers(Dong-Feng Biochemical Manufacture or Sigma) and calf thym us histone markers (Sigma) are used in parallel.…”

Section: Sds -P Olyacrylamide Gel Electrophoresis Of Proteinsmentioning

confidence: 99%

The effects of DNA intercalators on chromatin of chicken red blood cells --- differential extraction on nonhistone proteins

Wang

Zhu

1990

Cell Res

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Taking advantage of the effects on DNA secondary structure of two DNA-intercalators, ethidium bromide and chloroquine, we used each of them to treat nuclei from both mature erythrocytes and reticulocytes of chicken, as an alternative approach to study the relationships between DNA secondary structure, nuclear proteins and chromatin structure. We presented results of differential extraction of nuclear proteins from nuclei with DNA-intercalators , as well as preliminary characterizati on of these proteins. A 45kd protein is the major component in fractions extracted by both intercalators from nuclei from either mature erythrocytes or reticulocytes and seems to be a DNA-binding protein. Furthermore, from current concepts of functional aspects of DNA conformation and structural heterogeneity in chromatin and nuclear proteins , we have discussed both the significance of our resuits as well as technical aspects of this approach.

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“…29 Gels were run at pH 8. cm above the bottom of the gel. Gels were stained for protein in 0.04% Coomassie Brilliant Blue G 250 in perchloric acid 30 and destained in 7.5% acetic acid. Autoradiography of […”

Section: Methodsmentioning

confidence: 99%

Proteins binding to kallikrein and esterase A2 in the urine of salt-sensitive and salt-resistant rats.

Rapp¹,

Joseph²,

McPartland³

1982

Hypertension

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SUMMARY Iodine-labeled ([ I2S I]) rat urinary kallikrein and rat urinary TAME esterase A2 were used as probes to look for urinary and plasma proteins that bind to these enzymes. Such proteins are presumptive enzyme inhibitors. Complexes formed with labeled enzymes were identified by polyacrylamide gel electrophoresis followed by autoradiography. Urine from young (6 weeks old) Dahl saltsensitive (S) rats showed no, or only traces, of protein binding to kallikrein. Concomitant with the slow development of hypertension and proteinuria in S rats fed normal rat chow, one of the six kallikrein-binding proteins demonstrable in plasma was readily found in S-rat urine. This kallikreinbinding protein was called "KBP-1." R rats showed either no or much less KBP-1 in the urine, compared to S rats up to 5 months of age. A partly purified preparation of KBP-1 was shown to inhibit the TAME esterase activity of rat urinary kallikrein in the radiometric TAME assay. Urine of proteinuric S rats also contained two TAME esterase-binding proteins, TEBP-1 and TEBP-2, detected with the [ l25 I]-esterase A2 probe. As S rats aged from 3 to 8 months, free KBP-1 disappeared from the urine in spite of increased and marked proteinuria and the continued presence of KBP-1 in plasma. Concomitant with this age-related loss of urinary KBP-1 there was a marked shift in S urinary proteins binding to [ l25 I]-esterase A2 from TEBP-1 to TEBP-2. It was speculated that KBP-1 and TEBP-1 were the same protein detectable with either labeled kallikrein or labeled esterase A2. The concomitant disappearance of free KBP-1 (TEBP-1) and the appearance of free TEBP-2 in the urine of old, hypertensive, proteinuric S rats suggests that: 1) most of the KBP-1 (TEBP-1) is bound to enzyme{s) in old rats; or 2) KBP-1 (TEBP-1) is largely converted to TEBP-2 in old rats; or 3) both are true and that binding of KBP-1 (TEBP-1) to enzymes is associated with the generation of TEBP-2. (Hypertension 4:545-555, 1982) KEY WORDS • kallikrein • esteroprotease • hypertension • genetic hypertension • salt-sensitive rats W HEN Nustad and Pierce 1 isolated kallikrein from the urine of female rats in 1974, they also found an enzyme other than kallikrein that hydrolyzed the artificial substrate a-N-ptosyl-L-arginine methyl ester (TAME). The esterases were designated " A " and " B " in the order of elution from a DEAE ion exchange column; Nustad and Pierce showed that esterase B was rat urinary kallikrein.1 We 2 have recently described an additional TAME esterase that is present only in the urine of male rats. This enzyme was androgen-dependent and we called it "esterase A l . Received August 25, 1981; revision accepted December 7, 1981. was renamed "esterase A2" since it eluted from DEAE ion exchange chromatography after esterase Al. 2Urinary kallikrein is known to be decreased in human essential hypertension.3 " 7 Holland et al. 8 have shown that among essential hypertensives, those patients who had slight renal insufficiency also had reduced urinary kallikrein excretion, but tho...

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A procedure to increase the sensitivity of staining by Coomassie brilliant blue G250-perchloric acid solution

Cited by 184 publications

References 3 publications

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin

The effects of DNA intercalators on chromatin of chicken red blood cells --- differential extraction on nonhistone proteins

Proteins binding to kallikrein and esterase A2 in the urine of salt-sensitive and salt-resistant rats.

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