Purification of penicillinase (β-lactamase) and acid phosphatase from Staphylococcus aureus in one procedure

Schaeg, W; Bingöl, Recep; Blobel, H.

doi:10.1016/0005-2744(72)90351-8

Cited by 19 publications

(6 citation statements)

References 11 publications

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“…Although three studies have reported isoelectric points ranging from 8.7 to 9.55 for the type A 3-lactamase, details of the methods by which these values were determined were described in only the report by Schaeg et al (18,26,31). Our early attempts to perform isoelectric focusing on the staphylococcal P-lactamases involved the use of both impure P-lactamase preparations (i.e., concentrated broth supernatant) and standard techniques involving the use of nitrocefin-impregnated filter paper (19).…”

Section: Resultsmentioning

confidence: 99%

Characterization of four beta-lactamases produced by Staphylococcus aureus

Zygmunt

Stratton

Kernodle

1992

Antimicrob Agents Chemother

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Section: Resultsmentioning

confidence: 99%

Characterization of four beta-lactamases produced by Staphylococcus aureus

Zygmunt

Stratton

Kernodle

1992

Antimicrob Agents Chemother

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“…This was performed using the method described by SCHAEG et al (1972) in a 1lOml preparative isoelectric focusing column (Pharmacia-LKB) in 40 to 46 YO (w/v) sucrose density gradients with Ampholines of pH 3.5-10.0 (Pharmacia). The eluate of the ion exchange chromatography was mixed with both sucrose solutions, and the gradients were prepared with a gradient mixer (Pharmacia-LKB).…”

Section: Preparative Isoelectric Focusingmentioning

confidence: 99%

Purification and Further Characterization of a Haemolysin of Actinomyces pyogenes

Ding

Lämmler

1996

Journal of Veterinary Medicine, Series B

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Summary A haemolysin produced by Actinomyces pyogenes ATCC 8164 was purified from culture supernatant by ammonium sulphate and polyethylene glycol precipitation, ion‐exchange chromatography on DEAE‐Sephacel, and fast‐protein‐liquid‐chromatography on Superose 12 prep grade. The purified haemolysin, designated as pyolysin, displayed a single band on poly‐acrylamide gel electrophoresis, indicating a molecular weight of 55000. Additionally, using gel filtration, the same molecular weight was estimated. Further studies of the eluate of ion‐exchange chromatography using isoelectric focusing also revealed a single protein band at pH 9.38 with haemolytic activity. A specific antiserum produced against pyolysin inhibited the haemolytic activity. The purity of the isolated protein was also determined by Western Blot analysis with antiserum obtained from a cow inoculated with culture supernatant from A. pyogenes and Peptococcus indolicus. The isolated pyolysin appeared to be heat‐labile and displayed cytotoxic effects on poly‐morphonuclear leucocytes and on pTK2 kidney cells.

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“…Whereas the maximal Ki for meta-aminophenylbo- sorbed strongly to glass, sand, and the surfaces of negatively charged particles (26). He suggested that this binding resulted from the basic nature of staphylococcal ,B-lactamasethe best characterized variant, the type A r-lactamase, contains a high proportion of lysine residues (43 of 257 amino acids, or 16.7%) (1) and has a reported isoelectric point of 8.9 (28) to 9.55 (32). Prior to using the cation-exchange and affinity chromatographic protocol described in this report, we had employed sequential cation-exchange and gel filtration chromatography as described by Schaeg et al (32) to purify staphylococcal ,-lactamase.…”

Section: Discussionmentioning

confidence: 99%

“…He suggested that this binding resulted from the basic nature of staphylococcal ,B-lactamasethe best characterized variant, the type A r-lactamase, contains a high proportion of lysine residues (43 of 257 amino acids, or 16.7%) (1) and has a reported isoelectric point of 8.9 (28) to 9.55 (32). Prior to using the cation-exchange and affinity chromatographic protocol described in this report, we had employed sequential cation-exchange and gel filtration chromatography as described by Schaeg et al (32) to purify staphylococcal ,-lactamase. Although the type A P-lactamase from strain PC1 was purified in high yield, <1% of the type B ,B-lactamase was recovered from the gel filtration column (Sephadex G-100, superfine; Pha..nacia) by the same protocol (D.S.K.…”

Section: Discussionmentioning

confidence: 99%

“…aureus P-lactamase over the last 15 years are simpler than those originally described by Richmond, the majority still involve ion-exchange chromatography followed by either an additional ion-exchange chromatographic step (30) or gel filtration (1,20,32). Furthermore, since most of these modified protocols have been applied only to the purification of ,3-lactamase from strain PC1, whether they also can purify ,B-lactamase from other S. aureus strains, including those that produce type B, C, and D P-lactamases has not been determined.…”

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confidence: 99%

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Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography

Kernodle

Zygmunt

McGraw

et al. 1990

Antimicrob Agents Chemother

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Boronic acids are active-site inhibitors of serine P-lactamases, and a phenylboronic acid-agarose affinity column has been used to purify j-lactamase from crude cell extracts of several bacterial species. We applied phenylboronic acid-agarose chromatography to the purffication of Staphylococcus aureus P-lactamase. Two factors interfered with the success of the previously described single-step chromatographic protocol. First, staphylococcal ,l-lactamase exhibited non-active-site-mediated adsorption to the agarose used as a support for the meta-aminophenylborate ligand, preventing the recovery of j-lactamase from the column. Second, the staphylococcal (-lactamases exhibited low affinity for meta-aminophenylborate with inhibition constants (K's) ranging from 8.0 x 10-3 to 20.0 x 10-3 M. These problems were resolved by modifying the buffers utilized during chromatography and increasing the dimensions of the affinity column, and a two-stage procedure consisting of cation-exchange chromatography followed by affinity chromatography was used to purify each of the four variants of staphylococcal ,I-lactamase. The mean specffic activities of the purified type A, B, C, and D 3-lactamases were 44.6, 12.2, 10.6, and 30.8 ,3-lactamase in pure form from a variety of bacterial species, including Bacillus cereus, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella aerogenes, and Pseudomonas maltophilia (4). The present study investigates the applicability of the chromatographic method described by Cartwright and Waley to the purification of the four recognized variants of staphylococcal 1-lactamase. A modified protocol useful in the preparation of purified P-lactamase from S. aureus is described. MATERIALS AND METHODSOrganisms. S. aureus strains that produce each of the four variants of staphylococcal ,-lactamase were evaluated and included PC1(pl524) and RN11(pI258) (type A P-lactamase), 22260 and ST79M41 (type B P-lactamase), V137 (type C 1-lactamase), and FAR8 and FAR10 (type D P-lactamase).The sources and pedigrees of these strains have been described previously (12-14, 16, 23, 24, 26, 31

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Purification of penicillinase (β-lactamase) and acid phosphatase from Staphylococcus aureus in one procedure

Cited by 19 publications

References 11 publications

Characterization of four beta-lactamases produced by Staphylococcus aureus

Characterization of four beta-lactamases produced by Staphylococcus aureus

Purification and Further Characterization of a Haemolysin of Actinomyces pyogenes

Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography

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