John W. M. Mulders

X-ray structure of antistasin at 1.9 A resolution and its modelled complex with blood coagulation factor Xa.

The three‐dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 Å resolution by X‐ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55‐residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two β‐strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N‐terminal domain being wedge‐shaped and the C‐terminal domain flat. Docking studies suggest that differences in domain shape enable the N‐terminal, but not C‐terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N‐terminal domain of antistasin, comprising residues 15‐17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C‐terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain.

RECOMBINANT RAT FOLLICLE-STIMULATING HORMONE; PRODUCTION BY CHINESE HAMSTER OVARY CELLS, PURIFICATION AND FUNCTIONAL CHARACTERIZATION

In order to obtain homologous follicle-stimulating hormone (FSH) for in vivo and in vitro studies in the rat, rat recombinant (rec) FSH was produced in Chinese Hamster Ovary (CHO) cells. The synthesized rat recFSH was purified and subjected to physico-chemical and biological characterization. including a comparison with two rat pituitary (pit) and reference preparations (NIDDK-rFSH I-8 and NIDDK-rFSH-RP2) as well as with human recFSH (Org 32489). The molecular masses of rat recFSH and human recFSH were determined by SDS-polyacrylamide (SDS-PAGE) and were found to be similar, about 40 kD. The pI distribution of rat recFSH is similar to rat pitFSH, and slightly more acidic than human recFSH (3.6-5.6 vs 3.9-5.5, respectively) as determined by isoelectric focussing in immobilized pH gradients. Rat recFSH displayed dose-response curves parallel and in the same dose range as the rat pitFSH in receptor binding and in vitro bioassays. However, the in vivo activities of rat recFSH and rat pitFSH were 8824 and 3051 IU/mg, respectively, determined by the Steelman Pohley assay. Rat (pit and rec) and human FSH are very different. Human recFSH bound to both calf testicular membranes and CHO cells expressing the human FSH receptor (CHO hFSH-R) with about 10-fold higher affinity (Ka) than pituitary and recombinant rat FSH. In in vitro bioassays with immature rat Sertoli cells and CHO hFSH-R cells human recFSH was also about 10-fold more potent than the rat FSH preparations. In the in vitro bioassays with immature rat granulosa cells the difference was about 5-10-fold. These studies indicate that the receptor binding and in vitro activities of rat pitFSH and rat recFSH are similar. The differences in in vivo activity are probably due to the differences in glycosylation. The biological behaviour of rat FSH (pit and rec) is different from that of human FSH. Therefore, if the rat is used as a model for physiology of gonadotropic action, the results may be greatly influenced by the type (species) of hormone preparation used. The availability of homologous hormone preparations is therefore crucial.

Recombinant rat luteinizing hormone; production by Chinese hamster ovary cells, purification and functional characterization.

Rat recombinant (rec) luteinizing hormone (LH) was produced in Chinese hamster ovary (CHO) cells, to enable studies on LH physiology in this species with homologous hormone. The synthesized hormone was purified, and characterized physico-chemically and biologically in comparison with highly purified preparations of rat pituitary (pit) LH (NIDDK-rLH-I-7 and I-9) and to highly purified urinary (NIH, CR-121) and rec forms of human chorionic gonadotropin (hCG). The 33 kD molecular mass of rat recLH, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, was comparable with the 32 kD size of pitLH. In chromatofocusing, the isoforms of rat recLH distributed in the pI range 6.5-7.8, similar to rat pitLH. In receptor binding assays using rat testicular membranes, and physiologic salt concentration, rat recLH displayed a 5-10-fold higher affinity than rat pitLH, but about 100-fold lower affinity than hCG. In contrast, in low salt concentrations the affinities of rat recLH and rechCG to rat LH receptor were rather similar. The differences in potency in the mouse Leydig cell in vitro bioassay were in agreement with the receptor binding data at physiologic salt concentration. Neither rat recLH nor pitLH stimulated cAMP production or bound specifically to HEK 293 cells expressing the rec human LH receptor. When injected subcutaneously on four consecutive days to male rats (8.4-33.7 microg/rat/day) rat recLH did not induce seminal vesicle growth in comparison with a significant effect of human menopausal gonadotropin (hMG; 12.5-50 IU/rat/day). In contrast, ovulation was induced in 5/6 and 6/6 female rats following single injections of 3.75 and 7.5 microg of rat recLH, respectively, after pretreatment with 10 microg/kg of a GnRH-antagonist (Org 30850). In conclusion, rat recLH displays clearly lower in vivo and in vitro bioactivity than hCG. Nevertheless, it binds effectively to the rat LH receptor (with affinity dependent on salt concentration) and is bioactive in the mouse Leydig cell bioassay. This newly synthesized recombinant hormone provides a useful tool for further studies on the physiology of LH action in the rat, the most common animal model in reproduction research.

Do immunoassays differentially detect different acidity glycoforms of FSH

The possibility of the carbohydrate residues of glycoproteins affecting their recognition in immunoassays is an important and unresolved issue. This study looked for evidence of differential recognition of FSH glycoform preparations, of variable isoelectric point (pI) and known molarity, using three routine assays employing different antibody configurations.