Cytochrome c: immunofluorescent localization of the testis-specific form.

Mouse testes contain a unique form of cytochrome c. As demonstrated by the indirect immunofluorescence technique, the testis-specific cytochrome c is detectable in the primary spermatocyte and in cell types comprising the later stages of spermatogenesis. Interstitial cells, Sertoli cells, and spermatogonia contain the somatic form of cytochrome c, as does heart muscle.

Linkage relationships of six enzyme Loci in interspecific sunfish hybrids (genus lepomis).

Backcross hybrids produced from the bluegill, the red-ear sunfish, and their F(1) interspecific hybrid have been analyzed for the inheritance of six enzyme phenotypes. Malate dehydrogenase A and B, tetrazolium oxidase, 6-phosphogluconate dehydrogenase, skeletal muscle esterase, and liver alpha-glycerophosphate dehydrogenase are all inherited in a mendelian manner as codominant alleles at nuclear loci. 6-phosphogluconate dehydrogenase and alpha-glycerophosphate dehydrogenase are encoded by linked loci, undergoing recombination at a frequency of 15%-22%. No other case of linkage was observed. The absence of linkage between the homologous malate dehydrogenase loci is of particular interest. These interspecific hybrids appear to be very useful for studies of biochemical genetics.

Antigenic domains of the sperm-specific lactate dehydrogenase C4 isozyme.

Lactate dehydrogenase C4 (LDH-C4) is an antigenic protein that occurs only in spermatozoa and the mature testis. The antibody-combining sites of this enzyme were mapped by measuring the binding of anti-LDH-C4 by isolated peptides. Pure mouse LDH-C4 was digested with trypsin, and the resulting fragments were fractionated by reverse-phase high-pressure liquid chromatography. Rabbit anti-mouse LDH-C4 bound to 13 pure peptides. Amino acid compositions and partial or complete sequencing by the Edman degradation was used to identify eight of these fragments in the complete structure of the molecule. The relationship between structure and antigenicity of these peptides is discussed in detail. These data fit best to the domain model of protein antigenicity. This antigenic map of LDH-C4 will be useful in the design of a synthetic contraceptive vaccine.

Identification of an antigenic determinant of mouse lactate dehydrogenase C4.

A peptide bearing an antigenic determinant of the sperm-specific lactate dehydrogenase C4 isozyme (LDH-C4) has been isolated from a tryptic digest of the whole protein. This peptide, comprising residues 152-159 (MC152-159), reacts with rabbit anti-mouse LDH-C4. Immunization of rabbits with synthetic MC152-159 conjugated to bovine serum albumin induces an immune response which is specific for the peptide. Anti-MC152-159 IgG binds 125I-labeled mouse LDH-C4 and competition experiments demonstrate the specificity of this antigen antibody reaction.

The antigenicity of synthetic peptide fragments of lactate dehydrogenase C4.

Lactate dehydrogenase C4 (LDH-C4) is an antigenic protein found only in spermatozoa and the mature testis. Synthetic peptides containing the amino acid sequences of the C-subunit, designated MC5-15, MC97-110 and MC211-220, were each conjugated to the carrier proteins diphtheria toxoid or bovine serum albumin. Rabbits immunized with these peptide-carrier conjugates produced antibodies to the peptide that cross-reacted with native LDH-C4. These data support our map that identifies antigenic domains of LDH-C4. Such synthetic peptides will be useful in the design of a contraceptive vaccine.

Alternative mobile phases for enhanced chromatographic selectivity and increased sensitivity in peptide separations.

The standard method for separating peptide mixtures is reversed-phase high-performance liquid chromatography with gradients of increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. With modern instruments and columns, complex peptide mixtures can be separated, and low picomole amounts can be collected in tens of microliters. Difficult separations are addressed by modifying the gradient slope or organic eluant composition. Further improvements in resolution are often needed, requiring fundamental changes in mobile phase composition or selection of complementary chromatographic separation mechanisms. For the present study, tryptic digests of cytochrome c from various species were separated in the presence of dilute hydrochloric acid by reversed phase on a Waters Delta-PakTM C18 high-performance insert column and by strong cation exchange on a Waters Protein-PakTM SP 8HR. Different and enhanced reversed-phase selectivity was obtained by replacing trifluoroacetic acid with dilute hydrochloric acid at the same pH. The increased optical clarity of hydrochloric acid-based mobile phases in the low ultraviolet wavelengths yielded increased sensitivity. Very different selectivity was observed with the cation-exchange chromatography. These data expand the options for peptide mapping by providing additional selectivity combined with increased mass sensitivity and spectral information in the low ultraviolet.

Principles and practice of peptide analysis with capillary zone electrophoresis.

Capillary electrophoresis provides a new analytical tool that complements reversed-phase high-performance liquid chromatography separations of peptides. The principles of this technique and their application to developing peptide analyses are described. Detailed recipes for useful buffers and for preparation of the instrument are provided. A sequence of generally useful experiments is suggested. Basic troubleshooting guidelines are provided.

Reduction of fertility in female baboons immunized with lactate dehydrogenase C4.

Immunization of female rabbits and mice with the sperm-specific isozyme of lactate dehydrogenase, LDH-C4, significantly reduced their fertility. Similar studies have been extended to nonhuman primates. Two female baboons, immunized with human LDH-C4, produced low antibody titers. These titers were markedly enhanced by booster injections of murine LDH-C4. An additional seven female baboons responded with relatively high antibody titers after receiving murine LDH-C4 as both priming and booster dosages. All nine females received injections of murine LDH-C4 at varying times determined by serum titer levels during fertility studies. These antisera reacted with human, mouse, and baboon LDH-C4. In a series of breeding experiments, 22 of 30 matings, or 73%, were infertile as compared with 28% in control matings. This contraceptive effect of the vaccine containing LDH-C4 was related to antibody titer and was reversible. Normal pregnancies ensued in animals in which the titer declined after termination of booster injections of vaccine.

Capillary electrophoresis with electrospray mass spectrometry detection for low-molecular-mass compounds.

Mass spectrometry (MS) detection using electrospray ionization (ESI) has been explored for the separation by capillary electrophoresis (CE) of a number of sample mixtures containing low-molecular-mass species. Optimal sheath liquid composition has been determined using a peptide mixture in which femtomolar quantities of analyte were easily observed. Effects of CE buffer choice were studied in detail. Also, a separation of basic drugs in cough syrup has been successfully detected by ESI-MS. Using negative ionization, a mixture of alkyl sulfonates and a mixture of food dyes were analyzed. All components were easily resolved and identified by molecular weight.

Alternative mobile phases for enhanced chromatographic selectivity and increased sensitivity in peptide separations.

The standard method for separating peptide mixtures is reversed-phase high-performance liquid chromatography with gradients of increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. With modern instruments and columns, complex peptide mixtures can be separated, and low picomole amounts can be collected in tens of microliters. Difficult separations are addressed by modifying the gradient slope or organic eluant composition. Further improvements in resolution are often needed, requiring fundamental changes in mobile phase composition or selection of complementary chromatographic separation mechanisms. For the present study, tryptic digests of cytochrome c from various species were separated in the presence of dilute hydrochloric acid by reversed phase on a Waters Delta-Pak C18 high-performance insert column and by strong cation exchange on a Waters Protein-Pak SP 8HR. Different and enhanced reversed-phase selectivity was obtained by replacing trifluoroacetic acid with dilute hydrochloric acid at the same pH. The increased optical clarity of hydrochloric acid-based mobile phases in the low ultraviolet wavelengths yielded increased sensitivity. Very different selectivity was observed with the cation-exchange chromatography. These data expand the options for peptide mapping by providing additional selectivity combined with increased mass sensitivity and spectral information in the low ultraviolet.

Drug identification in biological matrices using capillary electrophoresis and chemometric software.

Drugs and their metabolites in biological specimens are analyzed by a variety of techniques. Capillary electrophoresis could provide another useful approach because of its unique selectivity and high resolving power. For routine use, however, rugged methods must be developed and combined with detection that confirms peak purity and identity in difficult sample matrices, such as, urine. In this study, capillary electrophoresis is used with diode array detection, and chemometric software is employed for spectral analysis. The software includes a series of chemometric tools. Principle Component Analysis and Iterative Target Transform Factor Analysis are used to inspect each electropherogram for spectral homogeneity of the peaks and to deconvolute comigrations. These algorithms are used to confirm the assay results. This approach is tested and demonstrated for the analysis of amphetamine and common interferences in human urine.

Optimization of high-performance liquid chromatographic peptide separations with alternative mobile and stationary phases.

Peptides are routinely separated with reversed-phase high-performance liquid chromatography using increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. While these separations may be improved by adjustments of gradient slope or substitutions of different solid-phase chemistries, many mixtures would benefit from systematic optimization of mobile phase components. Tryptic digests of cytochrome c from various species were separated on Waters Delta-Pak C18. The effects of varying pH as well as the concentration and type of ion-pair reagent were examined. In addition, low pH, ion-suppression/ion-pairing chromatography was inverted using a polymeric reversed-phase column at high pH with alkyl amine ion pairing. Finally, a tryptic digest of cytochrome c was resolved by ion-exchange chromatography with a strong cation-exchange high-performance liquid chromatography column. These data suggest a framework for dramatically changing the selectivity of peptide separations, leading to more satisfactory peptide mapping.

An allelic variant of the sperm-specific lactate dehydrogenase C4 (LDH-X) isozyme in humans.

An unusual pattern of LDH isozymes was observed by gel electrophoresis of an extract of a human testis. This isozyme composition is consistent with an allelic variant of Ldh-c.

Sperm-specific lactate dehydrogenase C4: antigenic structure and immunosuppression of fertility.

PIP: Isozyme study can provide useful information on structure-function relationships, biological specificity, gene regulation, and evolution. This paper reviews the isozyme lactate dehydrogenase (LDH)-C4, the product of a 3rd gene locus whose expression is restricted to the testis. A precise correlation between active spermatogenesis and LDH-C4 synthesis has been established, and the genes that encode sperm-specific enzymes have been shown to be under coordinate control. LDH-C4 does not appear until puberty and then is isolated from the immune system by the blood-testis barrier. The strong immune response to LDH-C4 and its unique genetic regulation and biological specificity suggest its potential as an alternative contraceptive technology. Humoral or cell-mediated immunity to the isozyme could have an adverse effect on sperm function, preventing fertilization. Female baboons immunized with LDH-C4 showed a substantial immune response as measured by circulating antibody titer and had 80% fewer offspring than nonimmunized animals when mated. The antibody is available along the route travelled by sperm and at the site of fertilization in the oviduct. Antibody in cervical mucus, uterine fluids, and oviducal fluids combines with LDH-C4 on the sperm surface and impedes the progress of the male gamete, presumably by agglutination. The immunosuppression of fertility appears to be related to the antibody titer and number of injections. The effect is reversible. These findings encourage further studies to develop LDH-C4 as a contraceptive vaccine. However, its efficacy must be increased and the natural product replaced with a snythetic antigen. Several small peptides bearing an antigenic determinant of LDH-C4 (e.g., MC 5-16, MC 152-159, MC 211-220, MC 101-115) have induced an immune response to mouse LDH-C4. Although the response is quantitatively less than that obtained after immunization with the natural protein, there is significant synthesis of specific antibodies. Further manipulations of the dosage, route of immunization, and schedule of these peptides are required to ensure maximum synthesis.^ieng

Binding of antibodies against antigenic domains of murine lactate dehydrogenase-C4 to human and mouse spermatozoa.

Peptide fragments of lactate dehydrogenase-C4 (LDH-C4) that contain antigenic sequences of the native protein have been identified. The present study describes the binding to murine and human spermatozoa of antibodies that were produced against synthetic peptides containing two of these sequences. Rabbits were immunized with peptides designated MC5-15 and MC211-220, conjugated to diphtheria toxoid (DT). Antisera from these rabbits were tested for binding to washed mouse epididymal sperm or human ejaculated spermatozoa using a solid-phase radioimmunoassay. Antisera bind to mouse sperm in this system at dilutions of 1:64,000. When these antisera are first absorbed with the native LDH-C4 molecule, significant inhibition of binding to sperm results. Antisera to both DT-MC5-15 and DT-MC211-220 bind to human sperm with similar but weaker patterns than seen with mouse sperm. These data indicate that the immune response to synthetic peptides containing antigenic sequences of LDH-C4 includes antibodies that specifically bind to this enzyme on the surface of sperm. In addition, there are shared antigenic sequences between mouse and human LDH-C4, including the MC5-15 and MC211-220 peptides.