scholarly journals Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations.

1983 ◽  
Vol 96 (2) ◽  
pp. 443-448 ◽  
Author(s):  
R K Carlin ◽  
D C Bartelt ◽  
P Siekevitz
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Protein ◽  
Cross Linking ◽  
Synaptic Membranes ◽  
Major Protein ◽  
Calmodulin Binding ◽  
Protease Digestion ◽  
Triton X ◽  
Peptide Maps ◽  
Proteolytic Product

A major protein of postsynaptic densities (PSDs), a doublet of 230,000 and 235,000 Mr that becomes enriched in PSDs after treatment of synaptic membranes with 0.5% Triton X-100, has been found to be identical to fodrin (Levine, J., and M. Willard, 1981, J. Cell Biol. 90:631) by the following criteria. The upper bands of the PSD doublet and purified fodrin (alpha-fodrin) were found to be identical since both bands (a) co-migrated on SDS gels, (b) reacted with antifodrin, (c) bound calmodulin, and (d) had identical peptide maps after Staphylococcus aureus protease digestion. The lower bands of the PSD doublet and of purified fodrin (beta-fodrin) were found to be identical since both bands co-migrated on SDS gels and both had identical peptide maps after S. aureus protease digestion. The binding of calmodulin to alpha-fodrin was confirmed by cross-linking azido-125I-calmodulin to fodrin before running the protein on SDS gels. No binding of calmodulin to beta-fodrin was observed with either the gel overlay or azido-calmodulin techniques. A second calmodulin binding protein in the PSD has been found to be the proteolytic product of alpha-fodrin. This band (140,000 Mr), which can be created by treating fodrin with chymotrypsin, both binds calmodulin and reacts with antifodrin.

scholarly journals Determination by photoaffinity labelling of the hydrophobic part of the binding site for acyl-CoA esters on acyl-CoA-binding protein from bovine liver

1990 ◽  
Vol 271 (1) ◽  
pp. 231-236 ◽  
Author(s):  
M Hach ◽  
S N Pedersen ◽  
T Börchers ◽  
P Højrup ◽  
J Knudsen
Keyword(s):  
Amino Acids ◽  
Staphylococcus Aureus ◽  
Binding Site ◽  
Binding Protein ◽  
Bovine Liver ◽  
Dodecanoic Acid ◽  
Reverse Phase ◽  
Hydrophobic Binding ◽  
The Difference ◽  
Peptide Maps

Acyl-CoA esters containing the photoreactive acids 12-(4′-azido-2′-nitrophenoxy)[1-14C]dodecanoic acid ([14C]AND-acid) or N-(4′-azido-2′-nitro-[3′-5′-3H]phenyl)-12-aminododecanoic acid ([3H]NANPA-acid) were synthesized. The photoreactive acyl-CoA esters could be bound to bovine acyl-CoA-binding protein (ACBP) and photocrosslinked to the protein. The photocrosslinked acyl-CoA-ACBP complex was separated from unlabelled ACBP on reverse-phase h.p.l.c. and the purified complex was digested with trypsin, Staphylococcus aureus V8 proteinase or endoproteinase Asp-N. By four independent peptide maps it was shown that the amino acids taking part in forming the hydrophobic binding site for acyl-CoA esters in bovine ACBP are located on the peptide segment from Asp21 to Asp38. Both photoreactive acyl-CoA esters used in this study labelled strongly in the segment from Tyr28 to Ala34. 12-(4′-Azido-2′-nitrophenoxy)[1-14C]-dodecanoyl-CoA ([14C]AND-CoA) also introduced a label at position Asp38, but o labelling was found before Ser29. In contrast, N-(4′-azido-2′-nitro[3′,5′-3H]phenyl)-12-aminododecanoyl-CoA [3H]NANPA-CoA) also labelled the segment from Asp21 to Tyr28. The difference in labelling by the two photoreactive ligands is most likely caused by different mobility of the arylazido group when linked to the fatty acid either through a phenolic O- or an anilinic N- bond.

scholarly journals Role of Penicillin-Binding Protein 2 (PBP2) in the Antibiotic Susceptibility and Cell Wall Cross-Linking of Staphylococcus aureus: Evidence for the Cooperative Functioning of PBP2, PBP4, and PBP2A

2005 ◽  
Vol 187 (5) ◽  
pp. 1815-1824 ◽  
Author(s):  
Tomasz A. Łęski ◽  
Alexander Tomasz
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Antimicrobial Agents ◽  
Binding Protein ◽  
Resistant Mutant ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
Experimental Proof

ABSTRACT Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.

scholarly journals A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions

1988 ◽  
Vol 255 (3) ◽  
pp. 999-1005 ◽  
Author(s):  
C Enrich ◽  
O Bachs ◽  
W H Evans
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Specific Protein ◽  
Subcellular Fractions ◽  
Calmodulin Binding ◽  
Overlay Technique ◽  
Protein Partitioning ◽  
Triton X

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol.

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

scholarly journals A ribosomal calmodulin-binding protein from Dictyostelium.

1991 ◽  
Vol 266 (34) ◽  
pp. 23091-23096
Author(s):  
J. Sonnemann ◽  
A. Bäuerle ◽  
T. Winckler ◽  
R. Mutzel
Keyword(s):  
Binding Protein ◽  
Calmodulin Binding
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