ABSTRACT
Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified.
Subject(s)
Membrane Proteins/isolation & purification , Plant Proteins/isolation & purification , Proteomics/methods , Cell Membrane/chemistry , Membrane Proteins/genetics , Musa/chemistry , Peptides/isolation & purification , Plant Proteins/genetics , Proteome/geneticsABSTRACT
The mechanism of proton pumping by P-type plasma membrane H(+)-ATPases is not well clarified. Site-directed mutagenesis studies suggest that Asp684, situated in transmembrane segment M6, is involved in coordination of proton(s) in plant plasma membrane H(+)-ATPase. This hypothesis is supported by atomic models of H(+)-ATPases built on the basis of the crystal structure of the related SERCA1a Ca(2+)-ATPase. However, more biochemical, genetic, and structural studies are required before we will be able to understand the nature of the proton binding site(s) in P-type H(+)-ATPases and the mechanism of action of these pumps.
Subject(s)
Cell Membrane/enzymology , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/metabolism , Amino Acid Sequence , Arabidopsis/enzymology , Calcium-Transporting ATPases/chemistry , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Sequence Alignment , Sequence Homology, Amino AcidSubject(s)
Arabidopsis/enzymology , Cell Membrane/enzymology , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/metabolism , Amino Acid Substitution , Arginine , Aspartic Acid , Binding Sites , Cloning, Molecular , Kinetics , Mutagenesis, Site-Directed , Protein Conformation , Protons , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymologyABSTRACT
The apoptosis-linked gene ALG-2 encodes a Ca(2+)-binding protein of the penta EF-hand family. To investigate the Ca(2+) binding properties of the recombinant ALG-2 protein, we have cloned ALG-2 cDNA from mouse liver mRNA. Sequence analysis showed that two types of clones were present. One (named ALG-2,5) corresponds to the published ALG-2 sequence (Vito, P., Lacana, E., and D'Adamio, L. (1996) Science 271, 521-525); the second (named ALG-2,1) is 6 nucleotides shorter, and the corresponding protein lacks the amino acid residues Gly(121) and Phe(122). Both transcripts are present in mouse tissues in the same 2:1 molar ratio. The ALG-2,5 and ALG-2,1 recombinant proteins are fully soluble in the metal-free form but can be precipitated from bacterial lysates by Ca(2+). In the presence of Tween the Ca(2+) binding profiles display two high affinity sites with [Ca(2+)](0.5) values of 1.2 and 3.1 microM for ALG-2,5 and ALG-2,1, respectively, plus one low affinity site. Using the yeast two-hybrid system we demonstrate that both proteins have a strong tendency to form homo- and heterodimers. In contrast to ALG-2, 5, the ALG-2,1 isoform does not interact with the target protein AIP-1, earlier described to play a role in apoptosis (Vito, P., Pellegrini, L., Guiet, C., and D'Adamio, L. (1999) J. Biol. Chem. 274, 1533-1540). We propose that the minor sequence difference between ALG-2,5 and ALG-2,1 affects the Ca(2+) binding properties and function of the proteins.
Subject(s)
Apoptosis , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calcium/metabolism , Genetic Variation , Liver/metabolism , Animals , Apoptosis Regulatory Proteins , Brain/metabolism , Cloning, Molecular , Female , Kidney/metabolism , Mice , Ovary/metabolism , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Secondary , RNA, Messenger/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Transcription, GeneticABSTRACT
A material of 100 patients aged 18-70 years, ASA groups 1-2, who were otherwise healthy with slight generalized sequelae of their illness but without limitations in their usual habits, who were admitted for gastroscopy were randomized to sedation with either diazepam or propofol. Prior to the examination, 0.2 mg/kg diazepam or 1 mg/kg propofol was administered. If necessary, this was supplemented with half of the initial dosage. No difference were found in the sedation. Patients in the propofol group remembered the surgeon's information significantly better and woke significantly more rapidly. Propofol caused significantly more pain on injection. We consider that propofol can be employed for outpatient gastroscopy.
