Energy metabolism and intracellular pH in boar spermatozoa.

The effect of energy metabolism on intracellular pH was studied in boar spermatozoa using nuclear magnetic resonance (NMR) spectroscopy and confocal microscopy with the pH-sensitive dye seminaphthorhodafluor (SNARF-1). Freshly ejaculated spermatozoa had a high adenylate energy charge (AEC=0.8), which decreased to 0.6 under aerobic conditions and to 0.2 under anaerobic conditions. Correspondingly, no ATP resonances but high AMP resonance were visible in (31)P-NMR-spectra of the spermatozoa. When an artificial oxygen buffer (Fluosol) and a purpose-built air supply system were used during (31)P-NMR data acquisition, ATP resonances reappeared whereas the AMP resonance disappeared. Boar spermatozoa kept under aerobic conditions have intracellular compartments that differ markedly in pH, as demonstrated by both (31)P-NMR spectroscopy and confocal microscopy. Using confocal microscopy, the midpiece of the flagellum in which all mitochondria are located was identified as an acidic compartment (pH(i-mp) 6.7). The intracellular pH of both the head (pH(i-h)) and the long principal piece of the flagellum (pH(i-pp)) were 7.2 and, thus, only slightly below the extracellular pH (pH(e) 7.3). Storage of spermatozoa in a glucose-free medium at 15 degrees C when they are immotile slowly shifted the pH(i-mp) from 6.7 to 6.9 within 20 h, whereas pH(i-h) and pH(i-pp) remained unchanged (pH 7.1-7.2). When glucose was present in the medium, all visible compartments of the spermatozoa as well as the medium were acidified to pH 6.2 within 20 h. Under these conditions a resonance at 4.8 mg kg(-1) appeared representing glycerol 3-phosphate.

Assignment of overlapping (1)H NMR signals in carp seminal plasma by proton-detected 2D C,H correlation spectroscopy.

The (1)H NMR spectrum of the perchloric acid extract of carp seminal plasma was heavily congested. It is demonstrated that proton-detected C,H chemical shift correlation spectroscopy (HSQC, HSQC-TOCSY) allows an unequivocal identification of proline, glutamate, taurine, and methionine sulfoxide, although several key proton signals were strongly overlapped.

Phosphorous metabolites in boar spermatozoa. Identification of AMP by multinuclear magnetic resonance.

Boar spermatozoa revealed three prominent resonances in the 31P-NMR spectrum of intact cells. Two of these are known to be GPC and Pi, the third is a phosphomononoester (PME), the identification of which was carried out by proton-detected 2D 1H,31P and 1H,13C chemical shift correlation experiments with gradient selection. The PME was unambiguously assigned to adenosine 5'-monophosphate (AMP). The identification was confirmed by an AMP consuming enzymatic assay. Other physiologically relevant PME's, in particular inosine 5-monophosphate (IMP) and sugar phosphates, were excluded. The intensity of the 31P signal of AMP in boar sperm extract was much higher than those of ADP and ATP, and in intact cells only AMP but no ATP was visible.

Nuclear magnetic resonance studies of boar seminal plasma. Problems encountered in the identification of small molecules: hypotaurine and carnitine.

Two major components in boar seminal plasma were assigned by 1H and 13C nuclear magnetic resonance spectroscopy. The first, previously called substance X (see Ref. 1, Biochim. Biophys. Acta 1243, 101-109 (1995)), was identified with difficulty as hypotaurine. This pointed to general difficulties in the NMR assignments of small molecules in mixtures of substances, even at the highest magnetic fields. In contrast, the identification of the second component as carnitine was obtained in a straightforward manner by total correlation spectroscopy and proton-detected 13C chemical shift correlation methods (gradient-selected heteronuclear single quantum coherence and heteronuclear multiple bond correlation). Carnitine is known as a transporter of fatty acids through membranes. Both compounds were ultimately confirmed by addition of the authentic compounds.

Spermatozoa: models for studying regulatory aspects of energy metabolism.

Spermatozoa are highly specialized cells, and they offer advantages for studying several basic aspects of metabolic control such as the role of adenosine triphosphate-(ATP)-homeostasis for cell function, the mechanisms of fatigue and metabolic depression, the metabolic channelling through the cytoplasm and the organization and regulation of glycolytic enzymes. Spermatozoa of four species with different reproductive modes are introduced and the first results are presented: Spermatozoa of the marine worm Arenicola marina are well adapted to external fertilization in sea water with fluctuating oxygen tension: they are motile for several hours in oxygen-free sea water, even when the ATP level is dramatically reduced. Anaerobic ATP production occurs by alanine, acetate and propionate fermentation probably by the same pathways known from somatic cells of this species. Under aerobic conditions the phosphagen system might function like a shuttle for energy-rich phosphate from mitochondria to the dynein-ATPases. Storage of turkey and carp spermatozoa for several hours without exogenous substrates and oxygen results in the degradation of phosphocreatine and ATP to inorganic phosphate and adenosine monophosphate (AMP), respectively. Despite low energy charges, stored spermatozoa of both species are capable of progressive movements. In carp spermatozoa fatigue of motility is not accompanied by the dramatic acidosis one discusses as an important effect in muscle fatigue. Energy metabolism of boar spermatozoa is typically based on glycolysis consuming extracellular carbohydrates and producing lactate and protons. The sperm seem to tolerate low intracellular pH (< 6.5). The lack of a phosphagen system (no energy shuttle from mitochondria to the distal dynein-ATPases) is probably compensated by a high glycolytic ATP-production in the mitochondria-free piece of the flagellum.

Multinuclear magnetic resonance studies of boar seminal plasma.

Multinuclear magnetic resonance studies were performed on aqueous solutions of lyophilisates of boar seminal plasma. 1H- and 13C-NMR spectral assignments were obtained by one- and two-dimensional experiments. Four prominent constituents were identified in the lyophilisate as well as in the original seminal plasma: inositol (95% myo-inositol, 5% scyllo-inositol), citrate, lactate and glycerophosphorylcholine (GPC). The concentrations of these compounds were evaluated from appropriate 1H- and 13C-NMR resonances using biochemically determined citrate as reference. 31P-NMR spectroscopy confirmed the presence of GPC and revealed phosphorylcholine, glycerophosphorylserine and glycerophosphorylethanolamine as further components.

Does a 2----5 beta-turn structure exist in enkephalins? Study of a fully protected Leu-enkephalin in organic solution by 17O-NMR.

The 17O chemical shifts of the Gly-2 and Gly-3 oxygens of a fully protected Leu-enkephalin were measured to be identical in acetone solution. This allows the conclusion that neither of these peptide oxygens is hydrogen bonded and that no specific 2----5 beta-turn structure exists to an appreciable extent.

Oxygen-17 relaxation times in the blood sera of rats with various cancers. Can a systemic effect be detected?

17O NMR relaxation times of water in the serum of rats with various cancers were measured. No systemic effect could be detected at 4.7 and 8.4 T. The serum T1(17O) value was 7.6 +/- 0.5 ms at 37 degrees C independent of the magnetic field. T2(17O) was approximately half T1(17O). The 17O relaxation times could be determined at a faster rate than the 1H relaxation times.

High-resolution 1H-NMR studies of monomeric melittin in aqueous solution.

High resolution 1H-NMR at 360 MHz was used to characterize monomeric melittin in aqueous solution. The monomeric form of melittin was found to prevail at 3 mM concentration, pH 3.0, and temperatures between 30 and 90 degrees C, both in the absence of salt and with 6 M guanidium chloride. From comparison with model peptides and studies of the effects of 6 M guanidium chloride and variable temperature on the NMR parameters it was concluded that monomeric melittin is predominantly in an extended flexible form, with the fragments 5--9 and 14--20 more highly structured than the rest of the amino acid sequence. The appearance of a second, low abundant form of monomeric melittin, which is in slow exchange on the NMR time scale with both the more abundant monomeric conformation and aggregated melittin, was attributed to cis-trans isomerism of the peptide bond Leu-13--Pro-14.

High-resolution 1H-NMR studies of self-aggregation of melittin in aqueous solution.

4 mM melittin solution in 0.05 M sodium phosphate buffer at p2H 7.0 and 30 degrees C was shown by ultracentrifugation to contain tetrameric melittin. Using the spectra of this species and the previously characterized monomeric melittin as references, high-resolution 1H-NMR at 360 MHz was used to investigate self-aggregation of melittin at variable temperatures, pH and ionic strength. The NMR parameters show that the spatial structure of aggregated melittin is different from monomeric mellitin in aqueous solution but resembles closely the conformation adopted by melittin bound to detergent micelles. Comparison of melittin bound to different detergent micelles and self-aggregated melittin in different aqueous media indicates that the mellitin monomers adopt similar conformations in all these systems. The present data suggest that melittin assumes an amphiphilic spatial structure which is stabilized both by the formation of mixed micelles with detergents or by self-aggregation.

Physicochemical studies of the protein-lipid interactions in melittin-containing micelles.

Complexes of melittin with detergents and phospholipids have been characterized by fluorescence, circular dichroism, ultracentrifugation, quasi-elastic light scattering and 1H nuclear magnetic resonance (NMR) experiments. By ultracentrifugation and quasi-elastic light-scattering measurements it is shown that melittin forms stoichiometrically well-defined complexes with dodecylphosphocholine micelles consisting of one melittin molecule and approximately forty detergent molecules. Evidence from fluorescence, circular dichroism and 1H nuclear magnetic resonance experiments indicates that the conformation of melittin bound to micelles of various detergents or of diheptanoyl phosphatidylcholine is largely independent of the type of lipid and furthermore appears to be quite closely related to the conformation of melittin bound to phosphatidylcholine bilayers. 1H NMR is used to investigate the conformation of micelle-bound melittin in more detail and to compare certain aspects of the melittin conformation in the micelles with the spatial structures of monomeric and self-aggregated tetrameric melittin in aqueous solution. The experience gained with this system demonstrates that high resolution NMR of complexes of membrane proteins with micelles provides a viable method for conformational studies of membrane proteins.

The 1H nuclear-magnetic-resonance spectra of Neurotoxin I and cardiotoxin Vii4 from Naja mossambica mossambica.

Two toxins from the venom of Naja mossambica mossambica, neurotoxin I and cardiotoxin VII4, were investigated in aqueous solution by high-resolution 1H nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization of the proteins included determination of the number of slowly exchanging amide protons which can be observed in 2H2O solution, measurement of the amide proton chemical shifts and exchange rates, characterization of the aromatic spin systems and the internal mobilities of aromatic rings, and studies of the pH dependence of the NMR spectra. For numerous resonances of labile and non-labile protons quite outstanding pH titration shifts were observed. It is suggested that these NMR parameters provide a useful basis for comparative structural studies of different proteins in the large group of homologous snake toxins. As a first application the NMR data presently available in the literature on neurotoxin II from Naja naja oxiana, toxin alpha from Naja nigricollis and erabutoxin a and b from Laticauda semifasciata have been used to compare these three proteins with neurotoxin I from Naja mossambica mossambica. This preliminary comparative study provides evidence that the same type of spatial structure prevails for these four homologous neurotoxins and that the folding of the backbone corresponds quite closely to that observed in the crystal structure of erabutoxin b. A second application is the comparison of cardiotoxin VII4 from Naja mossambica mossambica with the neurotoxins. The experimental data indicate that the folding of the polypeptide backbone is closely similar, but that the cardiotoxin molecule is markedly more flexible than the neurotoxins.