Chromium was proposed to be an essential element over 50 y ago and was shown to have therapeutic potential in treating the symptoms of type 2 diabetes; however, its mechanism of action at a molecular level is unknown. One chromium-binding biomolecule, low-molecular weight chromium-binding substance (LMWCr or chromodulin), has been found to be biologically active in in vitro assays and proposed as a potential candidate for the in vivo biologically active form of chromium. Characterization of the organic component of LMWCr has proven difficult. Treating bovine LMWCr with trifluoroacetic acid followed by purification on a graphite powder micro-column generates a heptapeptide fragment of LMWCr. The peptide sequence of the fragment was analyzed by MS and tandem MS (MS/MS and MS/MS/MS) using collision-induced dissociation and post-source decay. Two candidate sequences, pEEEEGDD and pEEEGEDD (where pE is pyroglutamate), were identified from the MS/MS experiments; additional tandem MS suggests the sequence is pEEEEGDD. The N-terminal glutamate residues explain the inability to sequence LMWCr by the Edman method. Langmuir isotherms and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to apoLMWCr. The sequence pEEEEGDD was found to bind 4 chromic ions per peptide with nearly identical cooperativity and binding constants to those of apoLMWCr. This work should lead to further studies elucidating or eliminating a potential role for LMWCr in treating the symptoms of type 2 diabetes and other conditions resulting from improper carbohydrate and lipid metabolism.
Laboratory experiments have been carried out to determine the bacterial plate counts on various meat preparation surfaces both before and after cleaning. The surfaces tested included wood, five proprietary cutting boards and formica. Experiments on both unused and scored surfaces showed that plate counts from wood were always greater than those from all the other boards tested indicating that the latter can be cleaned more efficiently, Tests were made to determine the incidence of salmonellae on wooden surfaces in frequent contact with raw meat. Ten (4.3%) of 235 samples of wood scrapings were found to contain salmonellae. These results confirm that wooden surfaces can be reservoirs and distributors of salmonellae.
The progressive neurodegenerative disease Friedreich's ataxia is caused by a decreased level of expression of frataxin, a putative iron chaperone. Frataxin is thought to transiently interact with ISU, the scaffold protein onto which iron-sulfur clusters are assembled, to deliver ferrous iron. Photoreactive heterotrifunctional chemical cross-linking confirmed the interaction between frataxin and ISU in the presence of iron and validated that transient interactions can be covalently trapped with this method. Because frataxin may participate in transient interactions with other mitochondrial proteins, this cross-linking approach may reveal new protein partners and pathways in which it interacts and help deduce direct, downstream consequences of its deficiency.
Transition metal-polyalanine complexes were analyzed in a high-capacity quadrupole ion trap after electrospray ionization. Polyalanines have no polar amino acid side chains to coordinate metal ions, thus allowing the effects metal ion interaction with the peptide backbone to be explored. Positive mode mass spectra produced from peptides mixed with salts of the first row transition metals Cr(III), Fe(II), Fe(III), Co(II), Ni(II), Cu(I), and Cu(II) yield singly and doubly charged metallated ions. These precursor ions undergo collision-induced dissociation (CID) to give almost exclusively metallated N-terminal product ions whose types and relative abundances depend on the identity of the transition metal. For example, Cr(III)-cationized peptides yield CID spectra that are complex and have several neutral losses, whereas Fe(III)-cationized peptides dissociate to give intense non-metallated products. The addition of Cu(II) shows the most promise for sequencing. Spectra obtained from the CID of singly and doubly charged Cu-heptaalanine ions, [M + Cu - H](+) and [M + Cu](2+) , are complimentary and together provide cleavage at every residue and no neutral losses. (This contrasts with [M + H](+) of heptaalanine, where CID does not provide backbone ions to sequence the first three residues.) Transition metal cationization produces abundant metallated a-ions by CID, unlike protonated peptides that produce primarily b- and y-ions. The prominence of metallated a-ions is interesting because they do not always form from b-ions. Tandem mass spectrometry on metallated (Met = metal) a- and b-ions indicate that [b(n) + Met - H](2+) lose CO to form [a(n) + Met - H](2+), mimicking protonated structures. In contrast, [a(n) + Met - H](2+) eliminate an amino acid residue to form [a(n-1) + Met - H](2+), which may be useful in sequencing.
One of the challenges for sequencing peptides by MS‐MS is the interpretation of complex fragment ion patterns. Many approaches have been explored seeking to produce fragmentation patterns that are more predictable and thus easier to interpret. One of these involves the formation of a gas‐phase adduct of peptide with a metal ion which acts to preferentially activate certain fragmentation pathways. In this month's Special Feature, Prof. Carolyn Cassady and co‐workers at the University of Alabama (Tuscaloosa, AL) examine the fragmentation patterns produced from first‐row transition metal adducts of some model peptides with the goal of gaining insight into the gas‐phase chemistry of transition metal‐cationized peptides and to increase the understanding of how specific transition metal ions affect dissociation of the peptide backbone.
The acclimation or adaptation of microorganisms to organic chemicals is an important factor influencing both the rate and the extent of biodegradation. In this study two acclimation procedures were evaluated in terms of their effectiveness in enhancing biodegradation, their relative ease of use in the laboratory, and the implications for biodegradability testing. In the single‐flask procedure, microorganisms were acclimated for 2 to 7 d in a single acclimation flask at constant or increasing concentrations of the test chemical without transfer of microorganisms. In the second procedure, the enrichment procedure, microorganisms were acclimated in a series of flasks over a 21‐d period by making adaptive transfers to increasing concentrations of the test chemical. Acclimated microorganisms from each procedure were used as the source of inoculum for subsequent biodegradation tests in which carbon dioxide evolution was measured. Six chemicals were tested: quinoline, p‐nitrophenol, N‐methylaniline, N,N‐dimethylaniline, acrylonitrile, and 2,2,4‐trimethyl‐1,3‐pentanediol monoisobutyrate. Microorganisms acclimated in the single‐flask procedure were much more effective than those acclimated in the enrichment procedure in degrading the test chemicals. The single‐flask procedure is more convenient to use, and it permits monitoring of the time needed for acclimation. The results from these studies have implications for the methodology used in biodegradation test systems and suggest caution before adopting a multiple‐flask, enrichment acclimation procedure before the performance of standardized tests for aerobic biodegradability.
Two biomolecules are known to bind chromium in the body: transferrin and low‐molecular‐weight chromium‐binding substance (LMWCr). While transferrin is responsible for the transfer of Cr from the bloodstream to the tissues, LMWCr has been proposed to have a function in the removal of chromium from the body and in the enhancement of insulin signaling. LMWCr is comprised of a peptide that can tightly bind four equivalents of Cr(III). While reports on the magnetic and spectroscopic properties of the chromic centers have appeared, research has been hampered by difficulties in characterizing the organic portion of the biomolecule. Treatment of LMWCr with trifluoroacetic acid releases the Cr, converts the amino terminal glutamate of the peptide to pyroglutamate, and generates a heptapeptide. This procedure has allowed the isolation of the chromium‐free peptide component of LMWCr from a variety of sources; this peptide can be sequenced by mass spectrometry.
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