ABSTRACT
Exposure to rotenone in vivo results in selective degeneration of dopaminergic neurons and development of neuropathologic features of Parkinson's disease (PD). As rotenone acts as an inhibitor of mitochondrial respiratory complex I, we employed oxidative lipidomics to assess oxidative metabolism of a mitochondria-specific phospholipid, cardiolipin (CL), in substantia nigra (SN) of exposed animals. We found a significant reduction in oxidizable polyunsaturated fatty acid (PUFA)-containing CL molecular species. We further revealed increased contents of mono-oxygenated CL species at late stages of the exposure. Notably, linoleic acid in sn-1 position was the major oxidation substrate yielding its mono-hydroxy- and epoxy-derivatives whereas more readily "oxidizable" fatty acid residues (arachidonic and docosahexaenoic acids) remained non-oxidized. Elevated levels of PUFA CLs were detected in plasma of rats exposed to rotenone. Characterization of oxidatively modified CL molecular species in SN and detection of PUFA-containing CL species in plasma may contribute to better understanding of the PD pathogenesis and lead to the development of new biomarkers of mitochondrial dysfunction associated with this disease.
Subject(s)
Cardiolipins/metabolism , Chromatography, High Pressure Liquid , Mass Spectrometry , Mitochondria/metabolism , Parkinsonian Disorders/metabolism , Rotenone , Substantia Nigra/metabolism , Animals , Arachidonic Acid/metabolism , Biomarkers/metabolism , Cardiolipins/blood , Disease Models, Animal , Docosahexaenoic Acids/metabolism , Linoleic Acid/metabolism , Male , Oxidation-Reduction , Parkinsonian Disorders/blood , Parkinsonian Disorders/chemically induced , Rats, Inbred Lew , Time FactorsABSTRACT
Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 microM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca(2+) uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca(2+) influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.
Subject(s)
Acrosin/antagonists & inhibitors , Prostatic Secretory Proteins , Proteins/pharmacology , Trypsin Inhibitors/pharmacology , Amino Acid Sequence , Animals , Cattle , Cell Membrane/metabolism , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/pharmacology , Glycoproteins , Guinea Pigs , Humans , Male , Mice , Molecular Sequence Data , Proteins/chemistry , Proteins/metabolism , Rats , Seminal Plasma Proteins , Sequence Homology, Amino Acid , Sperm Head/metabolism , Spermatozoa/drug effects , Spermatozoa/metabolism , Trypsin Inhibitor, Kazal Pancreatic , Trypsin Inhibitors/chemistryABSTRACT
A basic 54-kDa protein (pI approximately 8.8) that cross-reacts with anti-caltrin antisera has been detected and isolated by gel filtration and cation exchange chromatography from seminal vesicle content of the rat. The soluble protein spontaneously precipitated in NaHCO3-buffered solution at pH 7.8, but it was kept soluble in imidazole buffer containing EDTA and dithiothreitol at pH 7.0. In addition to the main band of 54 kDa, two faint immunoreactive fractions with molecular weights around 45,000 and 14,000 were also revealed by Western blotting. The presence of the rat caltrin sequence within the primary structure of the 54-kDa molecule has been investigated by sequencing the peptides generated by trypsin digestion. The sequence of the first 46 amino acid residues of rat caltrin has been found in one of the fragments produced by enzymatic cleavage. However, the exact location of the caltrin sequence in the whole 54-kDa protein has not been determined. The purified 54-kDa protein did not inhibit Ca2+ uptake by epididymal spermatozoa. Results indicated that this molecule represents an inactive precursor of caltrin and is enzymatically processed in the lumen of the seminal vesicle to the small and active calcium transport inhibitor protein. The immunoreactive proteins with intermediate molecular weights (45,000 and 14,000) could represent partially degraded products of the precursor. The lack of inhibitory activity of the precursor may be related to the molecule's having a size and conformation that would make it unable to interact with caltrin receptors on the sperm surface.
Subject(s)
Prostatic Secretory Proteins , Proteins/isolation & purification , Seminal Vesicles/chemistry , Testicular Hormones/isolation & purification , Amino Acid Sequence , Animals , Blotting, Western , Calcium/metabolism , Electrophoresis, Polyacrylamide Gel , Isoelectric Point , Male , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Proteins/chemistry , Proteins/pharmacology , Rats , Seminal Plasma Proteins , Spermatozoa/drug effects , Spermatozoa/metabolism , Testicular Hormones/chemistry , Testicular Hormones/pharmacology , Trypsin/metabolismABSTRACT
Caltrins, small basic proteins that inhibit calcium uptake by epididymal spermatozoa, have been purified from seminal vesicle content of the mouse and rat. Mouse caltrin (M(r) 8,476) contains 75 amino acid residues, 14 basic, 5 acidic, and 7 cysteines while rat caltrin (M(r) 6,217) has 56 residues, 10 basic, 5 acidic, and 6 cysteines; their pI values are 10.2 and 9.3, respectively. The proteins did not react with Ellman's reagent unless the cystine residues were previously reduced. The primary structures were determined by sequencing fragments generated by trypsin, clostripain, and endoproteinase Lys-C digestion. The sequences were ordered to give the total structural formula. The two molecules have no sequence similarity and are different from those of the bull and guinea pig previously reported. Only rat caltrin has a sequence of 13 residues nearly identical to that in guinea pig caltrin I. Both rat and mouse caltrin react with antibodies against bovine and guinea pig caltrins. Reduction and alkylation of cysteine residues suppressed the immunologic response of mouse caltrin; however, modified rat caltrin retained partially its immunoreactivity with the antiserum against guinea pig caltrin I. The same treatment abolished the calcium transport inhibitory activity of mouse caltrin and greatly reduced that of rat caltrin. It is likely that rat and mouse caltrins have the same physiological function as proposed for bovine caltrin; namely, to regulate the development of the Ca(2+)-dependent processes that "capacitate" sperm for fertilization.
