Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane

Fairbanks, Grant; Steck, Theodore L.; Wallach, Donald F. Hoelzl

doi:10.1021/bi00789a030

Cited by 8,425 publications

(2,980 citation statements)

References 49 publications

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“…Samples electrophoresed on 5% SDS gels [30] or agarose/acrylamide composite gels [31] were transferred onto Immobilon membrane and analysed for AF-28 epitope [17] or BC-3 epitope [32]. After blocking with 5% skim milk powder in PBS, the membranes were incubated with AF-28 antibody (1 : 1,000 dilution) or BC-3 antibody (1 : 1,000) in 0.5% skim milk powder in PBS for 1 h at room temperature then washed six times in buffer containing 0.1% tween-20 in PBS.…”

Section: Irnmunodetection With Monoclonal Antibodies Af-28 and Bc-3mentioning

confidence: 99%

Degradation of cartilage aggrecan by collagenase‐3 (MMP‐13)

Fosang

Knäuper²,

Murphy³

et al. 1996

FEBS Letters

345

217

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Section: Irnmunodetection With Monoclonal Antibodies Af-28 and Bc-3mentioning

confidence: 99%

Degradation of cartilage aggrecan by collagenase‐3 (MMP‐13)

Fosang

Knäuper²,

Murphy³

et al. 1996

FEBS Letters

345

217

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“…Molecular weight markers were myosin (205 kDa), β-galactosidase from E. coli (116 kDa), phosphorylase B from rabbit muscle (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa, a glycoprotein) and carbonic anhydrase from bovine erythrocytes (29 kDa). Gels were stained for protein by Coomassie blue and for glycoprotein using periodic acid and Schiff's reagent (Fairbanks et al, 1971). Analytical isoelectric focussing was performed using Ampholine PAG plates (LKB, Bromma, Sweden).…”

Section: Assaysmentioning

confidence: 99%

A xyloglucan oligosaccharide‐active, transglycosylating‐D‐glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings – purification, properties and characterization of a cDNA clone

et al. 1998

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“…Slab gel SDS-PAGE was performed as described by Laemmli (1970) using a Bio-Rad Mini-Protean I1 device. Gels were stained for protein with Coomassie Blue as described by Fairbanks et al (1971). Preparative SDS-PAGE was performed using a Bio-Rad model 491 prep cell following the manufacturer's protocols.…”

Section: Standard Proceduresmentioning

confidence: 99%

Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus

Solow¹,

Bischoff²,

Zylka³

et al. 1998

Protein Science

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When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [Y-'~P]ATP, several radiolabeled polypeptides were observed following SDS-PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35-kDa one, whose protein-phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35-and 50-kDa phosphoproteins were purified and their amino-terminal sequences determined. The former polypeptide's amino-terminal sequence closely matched a conserved portion of the a-subunit of succinyl-CoA synthetase, which forms an acid-labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50-kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro-Gly-ThrAsp/Ser-Gly-Val/Leu-Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6-phosphate to donate its phosphoryl group to the protein.Keywords: Archaea; Archaebacteria; hexosephosphate mutase; phosphoprotein; succinyl-CoA synthetase Phosphate represents one of nature's most versatile and important chemical moieties (Westheimer, 1987). It comprises (1) the basic unit for the cell's energy currency, ( 2 ) a key chemical activator of organic functional groups in biocatalysis, (3) a charge-rich anchor for confining biomolecules within the cell membrane, (4) the linker that holds together the polymers that encode genetic information, and (5) a molecular switch for modulating the functional properties of key proteins. Although our general appreciation of the importance of phosphate in living organisms extends to the members of the Archaea-the third, most recent, phylogenetic domain to be recognized (Olsen & Woese, 1993)-in many areas, specific information is lacking. This is particularly true with regard to the interactions of phosphate with archaeal proteins. Only recently have archaeal phosphoproteins begun to be identified. The first was a CheA-like histidine kinase that functions as part of a twocomponent signal transduction cascade in Halobacterium salinarium Reprint requests to: Peter J. Kennelly, Department of Biochemistry, Virginia Polytechnic Institute and State University, 123 Engel Hall, Blacksburg, Virginia 24061-0308; e-mail: pjkennel@vt.edu. (Rudolph & Oesterhelt, 1995). The second was a methyltransferase activation protein from Methanosarcina...

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Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane

Cited by 8,425 publications

References 49 publications

Degradation of cartilage aggrecan by collagenase‐3 (MMP‐13)

Degradation of cartilage aggrecan by collagenase‐3 (MMP‐13)

A xyloglucan oligosaccharide‐active, transglycosylating‐D‐glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings – purification, properties and characterization of a cDNA clone

Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus

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