Characterization of basic proteins of bull seminal plasma

We have employed high-performance liquid chromatography on reversed phase columns to analyse the major basic proteins from bull seminal plasma. The proteins were separated preparatively and characterized with respect to molecular mass, amino-acid composition as well as by means of immunodiffusion against specific antisera. The following proteins could be identified: bull seminal proteinase inhibitor II (BUSI II), two seminal RNAases, the seminal antimicrobial protein and proteolytic fragments, derived from it, and a hitherto unknown protein P6 of molecular mass 20 000 Da. Another unknown protein, P5, found to be formed during preparation of the basic protein fraction turned out to be a proteolytic fragment of protein P6 with a molecular mass of 8 750 Da for the polypeptide chain. Antisera against the isolated proteins were raised in rabbits and their specificity established. Single radial immunodiffusion was used to determine the concentration of the above basic proteins in bull seminal plasma: BUSI II (0.25 mg/ml), seminal RNAases (6.5 mg/ml) and protein P6 (2.9 mg/ml).     

Stimulation of bull seminal RNase by various basic proteins

The activity of purified bull seminal RNase was markedly stimulated by various basic proteins. At the half concentration of substrate RNA, basic proteins such as histones, high-mobility group chromosomal proteins and cytochrome c stimulated the enzyme activity 4-6 fold. Other non-basic proteins such as bovine serum albumin and human gamma-globulin were far less effective. In addition to enzyme-stimulating activity, basic proteins showed a marked enzyme-stabilizing activity, indicating the presence of a strong interaction between the enzyme and basic proteins.     

The major basic proteins of bull seminal vesicle secretion

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.     

Affinity chromatography of thyroxine-binding proteins from human serum

The affinity matrix prepared by the attachment of L-thyroxine (T4) to epichlorohydrine-activated Sepharose 4B biospecifically absorbs the T4-binding globulin (TBG), 25K and 80/27K proteins, immunoglobulin G (IgG) and albumin (HSA) from human normal and retroplacental sera. The absorbed protein patterns were shown to depend on the immobilized T4 concentration, pH, temperature and incubation time. The potent eluents desorbing 85-100% of the protein are 1 mM NaOH, 3 M NH4SCN, 10(-5) M T4 or 3 mM 8-anilinonaphthalene-1-sulfonic acid (ANS) for TBG; NaOH, NH4SCN, 3 mM MgCl2 or 12mM sodium cholate for 25K protein and HSA; NaOH, NH4SCN or MgCl2 for the 80/27K and 25K proteins and IgG. Moreover, T4 desorbs small amounts (6-8%) of the 80/27K and 25K proteins, while sodium cholate elutes about 6% of TBG. The eluted from T4-Sepharose 4B and further purified TBG, 25K and 80/27K proteins display different [125I]T4-binding activities within the pH range from 2 to 9 and differ by their resistance to thermal inactivation at 50-80 degrees C. Double radial immunodiffusion analysis with the use of antisera to TBG, 25K, 80/27K, HSA and IgG demonstrated that the proteins share no common antigenic determinants. It was concluded that the novel 25K and 80/27K proteins represent endogenous components of the human blood thyroid hormone-binding protein system rather than fragments or aggregates of the known T4-binding proteins.     

Affinity chromatography of nonhistone chromosomal proteins from lymphocytes on DNA-agarose columns

A two step procedure is presented consisting of hydroxyapatite and DNA-agarose chromatography which allows the isolation of nonhistone chromosomal proteins with different affinities towards single stranded DNA. The application of this fractionation scheme to nonhistone chromosomal proteins from bovine lymphocytes is described.     

Immunoreactive vasopressin in bull seminal fluid

Immunoreactive arginine vasopressin (irAVP) was measured in seminal fluid with and without extraction using a specific radioimmunoassay (RIA). A large fraction of irAVP was removed after extraction on octadecasilylsilica cartridges. The measured amount of irAVP corresponded to the levels found in blood plasma. Dilutions of seminal plasma extracts were parallel with the RIA standard curve. On reversed phase HPLC the extracted material coeluted with synthetic AVP. These findings suggest an identity of this immunoreactive material with intact AVP. During incubations of synthetic AVP and its analogue 8-D-arginine vasopressing (8-DAVP) in seminal plasma, immunoreactivity decreased considerably with the former peptide, while the concentration of 8-DAVP was not significantly altered.     

5'-nucleotidase from bull seminal plasma

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) occurs in bull seminal plasma in multiple forms. The heterogeneity does not reflect the existence of true isoenzymes, but is due to the association of the enzyme with particulate material and to molecular aggregation phenomena. Addition of detergents to native bull seminal plasma prevents molecular aggregation, solubilizes the particulate form of the enzyme, and results in the appearance of a single molecular form of the enzyme. Enzyme purification can be achieved after three chromatographic steps which involve negative adsorption of 5'-nucleotidase activity on DEAE-Sephadex A-50 followed by two affinity chromatographies on concanavalin A-Sepharose 4B and ADP-agarose. The enzyme appears to be a dimeric glycoprotein. Some properties of the enzyme, including substrate specificity and the effects of hydrogen ion concentration and of various divalent cations, are reported.     

Synthesis of seminal ribonuclease in isolated lobules of bull seminal vesicles

A procedure is described for preparing and maintaining in culture isolated lobules of bovine seminal vesicles, consisting of glandular acini, surrounded by little connective tissue and with free access to the external medium, in which secreted material can be collected. After 48 h in culture, the isolated lobules appeared indistinguishable, by morphological and biochemical criteria, from freshly isolated lobules. After much longer culture times about one third of the glandular cells were still capable of effective protein synthesis. Studying the biosynthesis of seminal ribonuclease with preparations of isolated lobules we found that the enzyme was synthesized and secreted; only the fully amidated isoenzyme was synthesized and secreted, indicating that production of the selectively deamidated isoenzymic forms occurred after secretion, newly synthesized protein was rapidly exported, indicating that the high levels of enzyme previously reported for the seminal vesicle tissue were essentially due to its content of stored secretion.     

Affinity chromatography of estrogen- and progesterone-binding proteins of human uterus

The purification of estrogen- and progesterone-binding proteins of human uterus by employing affinity resins coupled with steroid-bovine serum albumin conjugates, led to the isolation of preparations with estrogen- and progesterone-binding sites havingK _d values in the range of 0.96 to 1.20 × 10^-9 M. These were different from theK _d values of 10^-10 M and 10^-8 M obtained for two types of binding sites present in the crude cytosolic and nuclear fractions. The purified proteins sedimented on sucrose gradient withS values in the range of 3.6–4.4. The cytosolic and nuclear estrogen- and progesterone-binding proteins, thus purified, showed differences in specificity of binding to the hormone. While the cytoplasmic proteins were more specific in their binding to estradiol or progesterone, the nuclear proteins bound Cortisol with equal or moderate affnity. These results demonstrate the presence of distinct physiological forms of estrogen- and progesterone-binding proteins in the cytoplasm and nucleus, thus pointing to the importance of both these compartments in hormone action.     

Calcium-binding protein in bull seminal vesicle secretion and seminal plasma.

A protein which showed high affinity for calcium ions was isolated from bull seminal vesicle secretion and seminal plasma. Its calcium-binding activity depended on the ionic strength and pH of the medium. The dissociation constant was 7-7 X 10(-7) M and there were 14 binding sites per protein molecule. The molecular weight of calcium-binding protein from bull seminal vesicle secretion, estimated by the gel filtration method, was 110,000. The protein may be involved in the regulation of the calcium ion level in seminal plasma.     

Affinity chromatography of serum proteins using immobilized lectin from Vicia faba

When human serum is applied to a column of Sepharose-insolubilized lectin from Vicia faba, some serum proteins are bound which can be eluted by means of 0.1 M glucose solution. These proteins are parts of the immunoglobulins IgA, IgG, IgM, and the alpha2-macroglobulin. These particular types of serum protein are bound specifically, due perhaps to some structural variation in the carbohydrate moieties they contain.     

Stimulation of bull seminal Ca2+, Mg2+-dependent endonuclease by various DNA-binding proteins

We have measured ΔA transient absorption spectra in the Soret region and kinetics of photodissociation of oxymyoglobin (MbO₂) solutions following excitation by pulses of duration 350 fsec and 10 μJ energy at 307 nm. We observed an instantaneous bleaching of the absorbance at 414 nm and the appearance of a broad, red-shifted absorption band in the 438–470 nm region with a time constant of 250 fsec indicative of the formation of a short-lived deliganded Mb species which relaxes to the stable Mb with a constant of 3.5 psec. Following this early relaxation, changes in absorption kinetics indicate also a geminate recombination process of constant τ = 100 psec. These data demonstrate that the well established low quantum yield (φ = 0.03) of photodissociation in MbO₂ is related both to the relaxation of an excited Mb state and to a fast geminate recombination process.     

The effect of bull seminal ribonuclease on spermatogenesis in the rabbit

Bull seminal ribonuclease is a very strong antigen in rabbits. In most animals the antibodies were formed after the first course of immunizations. Antibodies were of IgG immunoglobulin character. When bull seminal ribonuclease itself, and in a complex with IgG immunoglobulin fraction, was injected into rabbit testes, lower weight testes and inhibition of seminiferous epithelium development were observed for a few weeks. Similar changes were evoked by subcutaneous injections of the enzyme. During the inhibition of spermatogenesis by the enzyme no histological changes in the intertubular tissue of testes or changes in androgen concentration in the blood serum were detected.     

Effect of bovine seminal plasma on neutrophil phagocytosis of bull spermatozoa

Seminal plasma was obtained from bulls of known fertility and was assessed for its effect on serum-induced phagocytosis of bull spermatozoa. A non-dialysable component was found to inhibit neutrophil phagocytic uptake of spermatozoa. The component was not destroyed by heating (56 degrees C for 30 min) or removed by ether. Use of a bactericidal assay confirmed the inhibition and suggested that inhibition does not permanently impair neutrophil function. Immunoperoxidase staining demonstrated the presence of bovine IgM, IgG1 and IgG2 on spermatozoa incubated in serum. Affinity of spermatozoa for the immunoglobulins was reduced when seminal plasma was added to the serum. These results suggest that bull seminal plasma can regulate phagocytic ingestion of spermatozoa. While the mechanism of this regulation remains obscure, it may be important in providing protection to spermatozoa immediately after ejaculation.