Task-related variation of postpharyngeal and cuticular hydrocarbon compositions in the ant Myrmicaria eumenoides.

In the ant Myrmicaria eumenoides we investigated postpharyngeal and cuticular hydrocarbons. At eclosion the glands contained almost no hydrocarbons and there were no lipid inclusions in the glandular epithelium. During the first 3 weeks of adult life the amount of hydrocarbons in the gland increased until day 5, and then remained constant while the lipid content in the epithelium increased steadily. Intracolonial hydrocarbon compositions were not uniform. Compositions of post-pharyngeal and cuticular hydrocarbons in individual ants varied simultaneously, but in different manner depending on the tasks of the ant (brood-tenders, foragers, scouts). Variations on the cuticle were greater than in the gland, but they were strongly correlated. Independent of ants' age and task, cuticular hydrocarbon compositions were dominated by alkenes and alkadienes. Task-specific differences in cuticular compositions were mainly in the amount of alkenes (high in foragers) and alkadienes (high in brood-tenders). Variation of hydrocarbons was low in ants up to 10 weeks old. Thereafter, ants fell into two groups: (1) ants that did not change their hydrocarbons and remained in the nest, and (2) ants that changed their hydrocarbon compositions and became foragers. These results contribute to an ongoing discussion of the dynamic relationship between post-pharyngeal and cuticular hydrocarbons.

Identification of Ser-1275 and Ser-1309 as autophosphorylation sites of the insulin receptor.

We have identified Ser-1275 and Ser-1309 as novel serine autophosphorylation sites by direct sequencing of HPLC-purified tryptic phosphopeptides of the histidine-tagged insulin receptor kinase IRKD-HIS. The corresponding peptides (Ser-1275, amino acids 1272-1292; Ser-1309, amino acids 1305-1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor beta-subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD-K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues.

cAMP-dependent phosphorylation of cytokeratin in hepatic inner mitochondrial membrane.

The phosphorylation pattern in mitochondrial fractions isolated from hepatocytes, preincubated with 32P-phosphate and stimulated with glucagon and calcium mobilizing hormones, was studied. Only in mitochondria from glucagon treated hepatocytes two phosphorylated protein bands were observed, one with a molecular weight (MW) of 54 kDa in the outer membrane fraction which, according to the literature, is suggested to represent protein kinase A; one with a MW of 20 kDa in the inner membrane fraction which has not been described earlier. Electroelution and digestion of the 20 kDa protein band yielded two tryptic peptides which were identified as fragments homologous to human cytokeratin type II (the sequence of rat cytokeratin type II is not known). From the amino acid composition and sequence, and from the known structure of type II cytokeratins, it is concluded that the 20 kDa phosphoprotein is composed of amino- and carboxylterminal proteolytic fragments of rat cytokeratin C8 which are tightly anchored in the inner mitochondrial membrane. The physiological significance of the possible interaction of cytoskeletal proteins with the mitochondrial inner membrane and its hormonal regulation are discussed.

Sequence analysis of lantibiotics: chemical derivatization procedures allow a fast access to complete Edman degradation.

Lantibiotics are antibiotic peptides produced via ribosomal synthesis of precursor proteins by gram-positive bacteria. They contain various unusual post-translational modifications, which include the formation of sulfide rings by lanthionine or beta-methyllanthionine, and 2,3-didehydroamino acids. The N-terminus may be blocked by a 2-oxobutyryl group and the C-terminus may be inaccessible in some of the lantibiotics. Due to these modifications the analysis of such peptides is very tedious. Chemical modifications using an ethanethiol-containing reaction mixture and/or trifluoroperacetic acid treatment were used to solve these analytical problems. Investigating the tetracyclic 22-peptide gallidermin and the N-terminally blocked tricyclic 34-peptide Pep5 as examples, a novel access to the primary structure of lantibiotics is demonstrated.

Strategies for nonradioactive methods in the localization of phosphorylated amino acids in proteins.

Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [32P]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.