Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity.

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome-c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain. Although the sequences of bovine and rat hemoglobin contain about 20% of amino acid substitutions, the families of structurally related peptides are very similar in both extracts. Several other proteins also give rise to identical or closely related peptide fragments in the two mammalian species. The outlined similarity extends almost exclusively to the most abundant peptides present in the extracts. The minor components show less overlap. Four hemoglobin-derived peptides isolated from rat brain were shown to be biologically active in tumor cells. Eleven are identical to bioactive peptides from other species. Ten structurally overlap with bioactive peptides from other sources. The data obtained show similar biosynthetic pathways of pool components in different species, the resultant peptides being aimed at fulfilling related functions.

LVV- and VV-hemorphins: comparative levels in rat tissues.

Screening of hemorphins in extracts of rat lung, brain, heart and spleen was carried out. The threshold for detection of hemorphins was 0.01 nmol for spleen and 0.05 nmol for other tissues. Both the content and the composition of hemorphins differed significantly in the tissues analyzed. Heart and lung extracts were rich in these peptides, the content of the most abundant components reaching 16-44 nmol/g of tissue. In contrast, spleen and brain contained much lower amounts of hemorphins, i.e. about 0.3-2.6 nmol/g of tissue. The most represented hemorphin in lung, heart and brain was VV-hemorphin-5, while the content of other members of the hemorphin family depended significantly on the tissue analyzed: lung extract was also rich in LVV-hemorphin-5, heart contained similar amounts of LVV-hemorphin-7 and LVV-hemorphin-5 and brain of LVV-hemorphin-6. In contrast, the hemorphin family in spleen was represented mainly by C-terminally shortened VV-hemorphins, i.e. VV-hemorphin-4 and VV-hemorphin-3. The levels of hemorphins in all cases were sufficient to activate the opioid receptors of the respective tissues.

Neokyotorphin and neokyotorphin (1-4): secretion by erythrocytes and regulation of tumor cell growth.

Human erythrocytes release neokyotorphin, the 137-141 fragment of hemoglobin alpha-chain into the supernatant of red blood cells primary culture. However, the neokyotorphin fragment 1-4 that is formed together with neokyotorphin inside the red blood cells and in various tissues is not found in the supernatant. Both neokyotorphin and its 1-4 fragment were shown to stimulate proliferation of L929 tumor cells.

Cytotoxic activity of acetylcholine receptor ligands.

Acetylcholine receptor ligands were studied for cytotoxicity in K562 human erythroid leukemia tumor cells. Cytotoxicity of carbachol, an agonist of acetylcholine receptors, atropine, an antagonist of muscarinic acetylcholine receptor, neurotoxin 11 (NT II) from Naja naja oxiana cobra venom and tubocurarine, antagonists of acetylcholine receptor of nicotinic type was exhibited in the 10-7-10-5 M concentration range. Several cytolytic processes, two for carbachol and three for other ligands, corresponding to different concentrations of each ligand were detected. All acetylcholine receptor ligands induced internucleosomal DNA fragmentation.

Different pathways of the release of cytotoxic proteins in LAK cells.

Human LAK cells were shown to release cytotoxic proteins by both Ca(2+)-dependent and Ca(2+)-independent mechanisms. CD3+ CD8+ CD16- and CD16+ CD8+ CD3- LAK cells were co-incubated with target cells in the presence of 4 mM EGTA. Although EGTA inhibited the exocytosis of cytolytic granules, supernatants obtained were cytotoxic for target cells. Cytotoxicity of CD3+ LAK cells and CD16+ LAK cells was due to cytotoxic proteins with MW 75 (p75), 35 (p35) and 22 (p22) kDa. LAK cells were also shown to release cytotoxic proteins by way of continuous secretion. After co-incubation in the absence of target cells LAK cells can secrete cytotoxic proteins with MW 75 (p75), 55 (p55), 38 (p38), 35 (p35), 25 (p25), 22 (p22) and 17 (p17) kDa.

Neokyotorphin and neokyotorphin (1-4): cytolytic activity and comparative levels in rat tissues.

The content of biologically active hemoglobin fragment neokyotorphin (TSKYR) as well as that of neokyotorphin fragment (1-4) (TSKY) were determined in extracts of lung, heart, and brain tissue of rats. The content of both peptides as well as the neokyotorphin/neokyotorphin(1-4) ratio differed significantly from each other in these tissues. The respective parameters deviate considerably from those of erythocytes where these peptides are originally formed. Comparative analysis of cytolytic activity of peptides was performed at human erythroid leukaemia (K562) and murine transformed fibroblast (L929) cell lines. TSKY showed reliable cytolytic activity in both cell lines, while neokytorphin was not cytotoxic. The data obtained lead to speculation that endogenous hemoglobin fragments might participate in regulation of tumor growth in vivo.