Mass spectrometric identification and quantification of hemorphins extracted from human adrenal and pheochromocytoma tissue.

The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the beta, gamma, delta, and epsilon chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.

Site-directed mutagenesis of recombinant human beta 2-glycoprotein I identifies a cluster of lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity.

beta2-Glycoprotein I (beta2GPI) is a phospholipid-binding serum protein with anticoagulant properties. It plays a vital role in the binding of anti-cardiolipin Abs purified from patients with autoimmune disease when assayed in a cardiolipin (CL) ELISA. Based on a three-dimensional model of beta2GPI, electrostatic calculations, and earlier peptide studies, a highly positively charged amino acid sequence, Lys282-Asn-Lys-Glu-Lys-Lys287, located in the fifth domain of beta2GPI, has been predicted to be the phospholipid binding site. We tested this hypothesis by site-directed mutagenesis of residues in the predicted phospholipid binding site and by assessing the mutants for phospholipid binding and anti-beta2GPI activity. A single amino acid change from Lys286 to Glu significantly decreased the binding of beta2GPI to CL. Double and triple mutants 2k (from Lys286, 287 to Glu286, 287), 2ka (from Lys284, 287 to Glu284, 287), and 3k (from Lys284, 286, 287 to Glu284, 286, 287) possessed no binding of Ab to beta2GPI in a CL ELISA, as well as no inhibitory activity on the binding of iodinated native beta2GPI to CL. These results indicate that the residues Lys284, Lys286, and Lys287 in the fifth domain of beta2GPI are critical for its binding to anionic phospholipids and its subsequent capture for binding of anti-beta2GPI Abs.

Chemical characterization of the parasitophorous vacuole membrane antigen QF 116 from Plasmodium falciparum.

On the basis of amino acid sequencing and immunological cross-reactivity, the Plasmodium falciparum parasitophorous vacuole antigens QF116 and exp-1/CRA are apparently identical. The epitope recognized by an inhibitory monoclonal antibody directed against QF116 is located proximal to the C-terminus of the protein. The QF116 protein is processed during maturation by the cleavage of a 22-amino-acid signal peptide and acylated as measured by labeling with myristic acid.

Isolation of inhibin alpha-subunit precursor proteins from bovine follicular fluid.

Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.

Immunologic control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus.

Cattle can be vaccinated against the tick Boophilus microplus by inducing an immunologic reaction against Ag in the tick gut. The uptake of antibody during feeding leads to severe damage to the parasite. One of the responsible tick gut Ag has now been purified and characterized: the Bm86 Ag. It is a membrane-bound glycoprotein present in very low abundance in extracts of partially engorged adult female ticks. It has an apparent m.w. of 89,000, an isoelectric point of 5.1 to 5.6 and an affinity for wheat germ lectin. Microgram amounts of this Ag are able to induce effective protection in cattle against the parasite, as shown by the decreased survival of ticks on vaccinated cattle and a reduction in engorgement weights and egg laying capacity of the survivors. Antisera to the Ag react with the surface of digest cells in the tick gut. As a result of the reaction with antibody, the endocytotic activity of these cells, which is a critical step in bloodmeal digestion in this tick, is strongly and rapidly inhibited. A number of peptides from this Ag, produced by digestion of the reduced and alkylated protein with endoproteinase lys-C, have been sequenced. One peptide has significant amino acid sequence homology with the epidermal growth factor precursor and a second peptide has homology with a putative protective antigen from Plasmodium falciparum.

The alpha-chain of murine CD8 lacks an invariant Ig-like disulfide bond but contains a unique intrachain loop instead.

The CD8 Ag is a cell surface heterodimer which demarcates predominantly cytotoxic T cells which are restricted by class I MHC Ag. The disulfide bonds within the murine structure were assigned in this study and the alpha-beta-interchain bond involves one or more cysteine residues located in each chain proximal to the plasma membrane or included within it. The location of the intrachain disulfide loop within the CD8 beta-chain confirms its proposed structural homology to an IgV domain but no corresponding disulfide loop is present within the alpha-chain. The invariant IgV disulfide loop has been replaced by a unique, short loop involving an unusual cysteine which is conserved in the CD8 alpha-chains of man, mouse, and rat. Despite its lack of precedent in other Ig-related structures, this unusual disulfide loop can be parsimoniously accommodated into a modified domain which has retained the major features of the Ig structural motif.