Flurbiprofen-Sepharose chromatography of the prostaglandin synthetase from bovine seminal vesicles

Flurbiprofen-Sepharose and Acetyl-Sepharose have been prepared by coupling dl-2-(2-fluoro-4-biphenylyl)propionic acid [Flurbiprofen] and acetic acid, respectively, to 3-(N-[3-aminopropyl])aminopropyl Sepharose 4B using a water soluble carbodiimide. The arachidonic acid oxygenase activity of solubilized bovine seminal vesicle microsomes is retarded during chromatography on Flurbiprofen-Sepharose but not Acetyl-Sepharose. Thus binding of the oxygenase to Flurbiprofen-Sepharose results from interaction with the immobilized inhibitor. However, the impure oxygenase is either not bound and/or not eluted in a biospecific manner since the abilities of flufenamic acid, R(+) and S(-)-5-cyclohexylindan-1-carnboxylic acid, and R and S-Naproxen to remove the enzyme from Flurbiprofen-Sepharose do not parallel the relative efficacies of these compounds as prostaglandin synthesis inhibitors. Nevertheless, gradient elution of arachidonic acid oxygenase activity from Flurbiprofen-Sepharose with flufenamic acid provides a 15 fold enrichment of the enzyme from solubilized bovine seminal vesicle microsomes in 80% yield indicating that this chromatographic reagent can be a powerful tool for use in purification of the prostaglandin synthetase.     

Biochemical properties of the prostaglandin/thromboxane synthetase of human blood platelets and comparison with the synthetase of bovine seminal vesicles

The enzyme system which synthesizes prostaglandins and thromboxanes in extracts of washed human platelets has been characterized with respect to kinetic parameters, pH and cofactor dependence, and inhibitor potencies. Arachidonate and dihomo-γ-linolenate were shown to be mutually competitive substrates, thus providing biochemical support for the assumption that both substrates are metabolized by the same cyclooxygenase, although they are ultimately metabolized to different patterns of products. Products of the synthetase of human leucocytes qualitatively resemble those obtained with human platelets. The prostaglandin synthetase of bovine seminal vesicles was studied under similar conditions, and kinetic parameters and inhibitor potencies were compared with those of platelet extracts.     

Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase)

An oligonucleotide probe was used to isolate a clone encoding prostaglandin endoperoxide synthetase (cyclooxygenase, EC 1.14.99.1) from a sheep seminal vesicle cDNA library. The protein predicted from nucleic acid sequence contains 599 amino acids including a 23-amino acid signal sequence. Thus, the mature cyclooxygenase deduced from the cDNA compares favorably in molecular size to the 70-kDa protein determined by gel electrophoresis. A putative transmembrane region and potential carbohydrate addition sites for N-linked sugars can be inferred from the amino acid sequence. Significantly, sequence similarities exist between cyclooxygenase, myeloperoxidase, and several other heme-containing proteins. The putative glycosylation sites, transmembrane domain, and sequence similarities with functionally related enzymes have been incorporated into a model for the topology of cyclooxygenase in the endoplasmic reticulum.     

Biochemical properties of the prostaglandin/thromboxane synthetase of human blood platelets and comparison with the synthetase of bovine seminal vesicles.

The enzyme system which synthesizes prostaglandins and thromboxanes in extracts of washed human platelets has been characterized with respect to kinetic parameters, pH and cofactor dependence, and inhibitor potencies. Arachidonate and dihomo-gamma-linolenate were shown to be mutually competitive substrates, thus providing biochemical support for the assumption that both substrates are metabolized by the same cyclooxygenase, although they are ultimately metabolized to different patterns of products. Products of the synthetase of human leucocytes qualitatively resemble those obtained with human platelets. The prostaglandin synthetase of bovine seminal vesicles was studied under similar conditions, and kinetic parameters and inhibitor potencies were compared with those of platelet extracts.     

Contraction of seminal vesicle and prostaglandin E1

It was studied how PGE₁ would affect the responses of isolated human seminal vesicles to adrenalin. PGE₁ in the final concentration of 1.3 μg/ml suppressed the contraction of human seminal vesicle that would have occurred in reaction to adrenalin added one minute later. When the concentration of PGE₁ was increased to 6.7 μg/ml, the inhibitory action was further enhanced. The meanings of this phenomenon were discussed.     

Stabilization of prostaglandin synthetase by immobilization of goat seminal microsomes on silica gel-G

Prostaglandin synthetase was immobilized by adsorption of goat vesicular microsomes on silica gel containing CaSO4 (silica gel G). Repeated cycles of enzymatic conversion of arachidonic acid to prostaglandin by the immobilized microsomes increased the product yield by 1.5 fold, in comparison to the same by free microsomal particles. The presence of Ca2+ in silica gel is responsible for this improved yield of prostaglandin as the divalent metal ion stabilized prostaglandin synthetase activity in a remarkable way. Microsomal particles immobilized on solid supports like alumina G and controlled pore glass were not very effective.     

Studies on the multiple forms of aminopeptidase A in bovine seminal vesicle secretion

After gel filtration, anion exchange chromatography and chromatofocusing aminopeptidase A (AP-A) of bovine seminal vesicle secretion (VS-S) was found to exist in multiple forms. Depending on the pH used (pH 6.5-8.5) gel filtration of VS-S revealed 1-3 forms of AP-A. At pH 8.5 two dissimilar low-molecular-weight forms of AP-A converted into aggregated high-molecular-weight form. The aggregated AP-A was dissociated into an intermediate form with Triton X-100 and/or sodium deoxycholate and further into two low-molecular-weight forms with thiol compounds and neuraminidase. The aggregated, intermediate and low-molecular-weight forms of AP-A displayed some differences in catalytic properties, modifier characteristics and thermal inhibition.     

Lack of covalent modification of prostaglandin synthetase (cyclo-oxygenase) by indomethacin

We have previously shown that aspirin irreversibly inhibits prostaglandin synthetase (cyclo-oxygenase) by acetylating the active site of the enzyme. By utilizing ¹⁴C-labeled indomethacin and a close analogue, we now show that indomethacin, unlike aspirin, does not covalently modify cyclo-oxygenase. Furthermore, indomethacin binding to the enzyme may be reversible since even though indomethacin can inhibit acetylation by aspirin, when enzyme inhibited by indomethacin (1 μM) is treated with 200 μM aspirin 3 times for 1 hour each, complete acetylation of cyclo-oxygenase is achieved.     

The influence of mating system on seminal vesicle variability among gobies (Teleostei, Gobiidae)

A variety of sexual selection mechanisms have been implicated to drive the variability of the male reproductive tract in internal fertilizers, while studies on external fertilizers have been largely limited to exploring the influence of sperm competition on testis size and sperm number. Males in the Gobiidae, a speciose teleost family of demersal spawners with external fertilization, are known to be characterized by accessory structures to the sperm duct called seminal vesicles. These seminal vesicles secrete a mucus-enriched seminal fluid. Seminal vesicle size and function have been demonstrated to be influenced by sperm competition at the intraspecific level. With the aim to test the factors influencing the development of these male organs at the interspecific level, an independent contrast analysis was performed on 12 species, differing in mating system type, sperm competition risk, and duration of egg deposition. The type of mating system appears to be the main factor significantly affecting development of seminal vesicles, with males of monogamous species completely lacking or having extremely reduced organs.     

Ultrastructure and function of the seminal vesicle of Bittacidae (Insecta: Mecoptera)

The fine structure of the seminal vesicle and reproductive accessory glands was investigated in Bittacidae of Mecoptera using light and transmission electron microscopy. The male reproductive system of Bittacidae mainly consists of a pair of testes, a pair of vasa deferentia, and an ejaculatory sac. The vas deferens is greatly expanded for its middle and medio-posterior parts to form a well-developed seminal vesicle. The seminal vesicle is composed of layers of developed muscles and a mono-layered epithelium surrounding the small central lumen. The epithelium is rich in rough endoplasmic reticulum and mitochondria, and secretes vesicles and granules into the central lumen by merocrine mechanisms. A pair of elongate mesodermal accessory glands opens into the lateral side of the seminal vesicles. The accessory glands are similar to the seminal vesicle in structure, also consisting of layers of muscle fibres and a mono-layered elongated epithelium, the cells of which contain numerous cisterns of rough endoplasmic reticulum and mitochondria, and a few Golgi complexes. The epithelial cells of accessory glands extrude secretions via apocrine and merocrine processes. The seminal vesicles mainly serve the function of secretion rather than temporarily storing spermatozoa. The sperm instead are temporarily stored in the epididymis, the greatly coiled distal portion of the vas deferens.     

Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (3) induction of hyaluronic acid synthetase by prostaglandin F2alpha.

The mechanism of the stimulatory effect of prostaglandin(PG)F2alpha on the production of hexosamine-containing substances by cultured fibroblasts was studied. Treatment of the cells with 1 microgram/ml of PGF2alpha resulted in a doubled net synthesis of acidic glycosaminoglycans during 20 hrs measured with uronic acid as index, and also resulted in 300 per cent increase of 3H-glucosamine incorporation into hexosamine-containing substances during the first 6 hrs. Fractionation of the PGF2alpha-stimulated hexosamine-containing substances with double isotope technique revealed that hyaluronic acid was the most stimulated component. Prior to the increase of hyaluronic acid, hyaluronic acid synthetase activity was found to be augmented by PGF2alpha as high as 4 times over the control. The augmentation of hyaluronic acid synthetase activity by PGF2alpha did not take place if actinomycin D was simultaneously present in the culture medium, suggesting that PGF2alpha induced the enzyme.