Porcine follicular fluid contains several low molecular weight inhibitors of follicle-stimulating hormone binding to receptor

Porcine follicular fluid (PFF) inhibited the binding of 125I-human follicle-stimulating hormone (hFSH) to receptor in vitro in a dose-dependent fashion. PFF (2.5 l) was fractionated on the basis of apparent molecular weight (Mr) by ultrafiltration using hollow fibers and membranes of precalibrated pore size. Desalted, low Mr (500-5000) subfractions containing FSH-binding inhibitor (FSH-BI) activity were further purified by Sephadex G10 gel filtration and anion-exchange high-performance liquid chromatography (HPLC). This resulted in the partial purification of several low Mr FSH-BIs. Three major peaks of FSH-BI were resolved on the Sephadex G10 column eluted with water; G10-1 [elution volume (Ve)/exclusion volume (Vo) = 1.1] had only FSH-BI activity, while G10-2 (Ve/Vo = 1.4) and G10-3 (Ve/Vo = 1.5) had both FSH-BI and luteinizing hormone (LH)-BI activities. A fourth strongly retarded peak (G10-4; Ve/Vo = 2.7) was also obtained. This latter fraction had only FSH-BI activity and represented less than 1% of the FSH-BI activity applied to the column. No separation of these fractions was obtained when the column was eluted with 10 mM ammonium acetate instead of water, suggesting resolution was due to ion-exchange or hydrophobic interactions with the Sephadex. Anion-exchange (Polyanion SI) HPLC of G10-1, G10-2 or G10-3 samples resolved several fractions with FSH-BI activity. A fraction unretained at either pH 5.0 or 7.0 (HPLC-1) was present in all samples. A fraction strongly retained by the column (HPLC-2) and a fraction eluted between 0.13 to 0.24 M acetate (HPLC-3) were present in G10-1 and G10-2 but not in G10-3. HPLC-4, eluted between 0.32 to 0.36 M acetate at pH 5.0, was detected only in G10-3 samples. The most potent low Mr FSH-BI obtained (HPLC-2) inhibited FSH binding by 50% at a dose of 10 micrograms and was enriched approximately 2500-fold relative to whole follicular fluid. These results indicate that PFF contains several low (500-5000) Mr inhibitors of FSH binding to receptor in vitro which differ on the basis of charge, hormone specificity and possibly molecular size and hydrophobicity.     

The binding of low molecular weight heparin to hemostatic enzymes

A low molecular weight preparation of porcine heparin (specific anticoagulation activity = 125 units/mg) was fractionated to obtain a mucopolysaccharide product of 6500 daltons (specific anticoagulant activity = 373 units/mg) that is homogeneous with respect to its interaction with antithrombin. This material was treated with fluorescamine in order to introduce a fluorescent tag into the mucopolysaccharide. Initially, we showed that the fluorescamine-heparin conjugate and the unlabeled mucopolysaccharide interacted with antithrombin in a virtually identical fashion. Subsequently, we demonstrated that labeled heparin could be utilized in conjunction with fluorescence polarization spectroscopy to monitor the binding of mucopolysaccharide to thrombin, factor IXa, factor Xa, and plasmin. The interaction of this complex carbohydrate with thrombin exhibited a stoichiometry of 2:1 with KH1T DISS = KH2T DISS = 8 x 10(-7) M. The formation of mucopolysaccharide . factor IXa complex is characterized by a stoichiometry of 1:1 with KHIXa DISS = 2.58 x 10(-7) M. The binding of heparin to factor Xa or plasmin occurred with low avidity. Therefore, the stoichiometries of these processes could not be established. However, our experimental data were compatible with a single-site binding residue with KHXa DISS = 8.73 x 10(-6) M and KHPL DISS = approximately 1 x 10(-4) M, respectively.     

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin (Mr 6500) in the presence of alpha 1-proteinase inhibitor (Mr 54,000), ovoinhibitor (Mr 50,000) and ovomucoid (Mr 27,000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.     

Muscarinic cholinergic binding sites on intact human lymphocytes

Using the muscarinic chalinergic ligand [³H]-quinuclidinyl benzilate, we have demonstrated that intact, viable human lymphocytes possess specific muscarinic binding sites. The binding is saturable, proportional to cell number, and is displaceable by atropine, benztropine, trihexyphenidyl and scopolamine. The apparent kd is 67 nM and the number of binding sites per cell is on the order of 5 × 10⁴. Not only do these findings provide a pharmacological basis for the observed effects of muscarinic agents on lymphocyte function, they also demonstrate the utility of human peripheral blood lymphocytes for investigation of abnormalities of the muscarinic cholinergic system.     

Antithrombin binding of low molecular weight heparins and inhibition of factor Xa

Fluorescence and stopped flow methods were used to compare clinically used heparins with regard to their ability to bind to antithrombin and to accelerate the inactivation of factor Xa. Titration of antithrombin with both low molecular weight heparin (LMWH) (enoxaparin, fragmin and ardeparin) and unfractionated heparin (UFH) produced an equivalent fluorescence increase and indicates similar affinity of all heparin preparations to antithrombin. However, relative to UFH enoxaparin, the LMWH with the smallest average molecular mass, contained only 12% material with high affinity for antithrombin. The rate of factor Xa inhibition by antithrombin increased with the concentration of the examined heparins to the same limiting value, but the concentration required for maximal acceleration depended on the preparation. According to these data the high affinity fraction of the heparin preparations increased the intrinsic fluorescence and inhibitory activity equally without additional effects by variations in chain length and chemical composition. In contrast, in the presence of Ca UFH accelerated the inhibition of factor Xa by antithrombin 10-fold more efficiently than comparable concentrations of the high affinity fractions of enoxaparin and fragmin. The bell-shaped dependence of this accelerating effect suggests simultaneous binding of both proteins to heparin. In conclusion, under physiologic conditions the anti-factor Xa activity of heparin results from a composite effect of chain length and the content of material with high affinity to antithrombin. Thus, the reduced antithrombotic activity of LMWH relative to UFH results from a smaller content of high affinity material and the absence of a stimulating effect of calcium.     

Isolation of a low molecular weight zinc binding ligand from human milk

A low molecular weight zinc binding compound from human milk has been purified by ultrafiltration, gel filtration, and ion-exchange chromatography. Evidence is provided that this compound is citrate. A higher amount of citrate-bound zinc was found in human milk than in cow's milk. It is suggested that the therapeutic value of human milk for patients with the genetic disorder of zinc metabolism acrodermatitis enteropathica (AE) derives from a greater content of bioavailable zinc citrate in human than in cow's milk.     

Receptor-bound uPA is reversibly protected from inhibition by low molecular weight inhibitors

Urokinase-type plasminogen activator (uPA) plays a ubiquitous role in cell migration and invasiveness. Amiloride, a competitive inhibitor of uPA, can inhibit endothelial cell (EC) outgrowth during angiogenesis. To address the question of whether amiloride blocked angiogenesis by inhibiting uPA, we undertook a study of uPA expression in sprouting EC in vitro and the effects of amiloride on both enzymatic and morphogenetic activity. As expected, amiloride inhibited soluble uPA (suPA) with an IC(50) of 45-85 microm, however, receptor-bound uPA (rbuPA) from the sprouting EC was insensitive to amiloride. Removal of uPA from its receptors confers sensitivity to inhibition by amiloride suggesting that a reversible conformational change may mediate the insensitivity of rbuPA to amiloride and its analogs. In summary, we found no evidence to support the hypothesis that amiloride blocks capillary outgrowth by inhibition of uPA, but were able to successfully demonstrate a functional difference between two physiological forms of this important matrix-degrading enzyme.     

Discovery of novel low molecular weight inhibitors of IMPDH via virtual needle screening

Novel, low molecular weight inhibitors of IMPDH have been discovered through the application of a validated virtual screening protocol. A series of 21 IMPDH inhibitors were used to validate the docking procedure. Application of this procedure to the selection of compounds for screening from an in-house database resulted in a 50-fold reduction in the size of the screening set (3425 to 74 compounds) and gave a hit-rate of 10% on biological evaluation.     

Characterization of the binding of alpha-bungarotoxin to bacterially expressed cholinergic binding sites

Bacterially expressed cDNA fragments of the alpha-subunit of the nicotinic acetylcholine receptor previously have been shown to bind alpha-bungarotoxin (Gershoni, J. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4318-4321). Here, a novel system has been developed in which totally synthetic alpha-bungarotoxin binding sites are expressed in Escherichia coli transformants. The amino acid sequences, alpha 184-200 and alpha 184-196 of the Torpedo californica alpha-subunit of the nicotinic acetylcholine receptor were expressed as trpE fusion proteins via the expression vector pATH2 and a method for the enrichment of these fusion proteins is described. Quantitative analysis of toxin binding to the recombinant binding sites demonstrates that they bind toxin with affinities of KD = 2.5 X 10(-7) and 4.7 X 10(-6) M, respectively. Furthermore, the pharmacological profile of alpha 184-200 qualitatively reflects that of the intact receptor. These data not only indicate that the area of alpha 184-200 is an essential element of the cholinergic binding site but that residues alpha 197-200 contribute a point of contact between the receptor and alpha-bungarotoxin.     

Identification of new inhibitors for low molecular weight protein tyrosine phosphatase isoform B

The National Cancer Institute Diversity Set II (1356 compounds) and Diversity Set III (1597 compounds) were screened via in silico methods as potential inhibitors of low molecular weight protein tyrosine phosphatase (LWM-PTP) isoform B (EC 3.1.3.48). Those candidates that demonstrated comparable or better docking scores than that of pyridoxal 5′-phosphate (PLP), one of the most potent known inhibitors of LMW-PTP with a competitive inhibitor dissociation constant (K_is) of 7.6 μM (pH 5.0), were analyzed via in vitro kinetic assays against LMW-PTP isoform B. While none of the compounds tested in vitro was significantly better that PLP, five compounds showed comparable inhibition. These five compounds are very diverse in structure and represent new therapeutic leads for inhibition of this isozyme.     

Virtual screening of low molecular weight mushrooms compounds as potential Mdm2 inhibitors

In some human cancer cases, the activity of p53 is inhibited by over-expressed Mdm2. The Mdm2 acts as an ubiquitin ligase, resulting in p53 ubiquitination and subsequent p53 proteasomal degradation. The disruption of the Mdm2-p53 interaction using small-molecule inhibitors is recognized as a promising strategy for anti-cancer drug design. Mushrooms are an important source of powerful compounds with anti-tumour properties. In this study, the first virtual screening of low molecular weight compounds present in mushroom is presented as potential Mdm2 inhibitors. A re-docking and cross-docking method was used to validate the virtual screening protocol. The steroids: ganoderic acids X (K_i?=?16nM), Y (K_i?=?22nM) and F (K_i?=?69nM); 5,6-epoxy-24(R)-methylcholesta-7,22-dien-3β-ol (K_i?=?74nM) and polyporenic acid C (K_i?=?59nM) stand out as the top ranked potential inhibitors of Mdm2. The docking pose of the most promising compounds were carefully analysed and the information provided shows several interesting starting points for further development of Mdm2 inhibitors.     

The binding characteristics of cholinergic sites in rabbit spermatozoa

Binding of neurotrophic ligands to rabbit spermatozoa was studied. Nicotinic cholinergic antagonists, [3H]alpha-bungarotoxin and [3H]dihydro-beta-erythroidine (DE), bound with high affinity to different sites in the tails of rabbit spermatozoa with the former binding to 10,207 sites/cell and the latter to 562 sites/cell. alpha-Bungarotoxin and DE sites resemble nicotinic sites in brain in binding affinity and specificity. [3H]Quinuclidinyl benzilate (QNB), a muscarinic cholinergic antagonist, also bound with high affinity to a single class of sites located in the heads and tails of rabbit spermatozoa. The binding characteristics of the sperm muscarinic site are similar to muscarinic sites in both innervated and noninnervated cells. Rabbit spermatozoa incubated for 16-18 h in a medium which supported motility for an extended period possessed fewer binding sites than nonincubated spermatozoa for [3H] alpha-bungarotoxin and [3H]QNB and the KD for the latter ligand was also lower. Ligands specific for the kappa and delta opiate receptors showed no affinity for rabbit spermatozoa.     

Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments

A set of 11 clones encoding putative GTP binding proteins highly homologous to the yeast YPT1/SEC4 gene products have been isolated from an MDCK cell cDNA library. We localized three of the corresponding proteins in mammalian cells by using affinity-purified antibodies in immunofluorescence and immunoelectron microscopy studies. One, the MDCK homolog of rab2, is associated with a structure having the characteristics of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus. The second, rab5, is located at the cytoplasmic surface of the plasma membrane and on early endosomes, while the third, rab7, is found on late endosomes. These findings provide evidence that members of the YPT1/SEC4 subfamily of GTP binding proteins are localized to specific exocytic and endocytic subcompartments in mammalian cells.     

In situ molecular weight determination of rat brain [3H]phenytoin binding sites

Using irradiation inactivation analysis of specific [3H]phenytoin binding to rat brain we have demonstrated that there are two different binding sites involved, with molecular weights of 73238 +/- 1535 (higher affinity site) and 108121 +/- 6935 (lower affinity site).     

A low molecular weight colchicine binding protein from the aquatic Phycomycete Allomyces neo-moniliformis

Summary A colchicine binding protein of the zoospore, flagellum, and pre- and post-mitotic germlings of the aquatic Phycomycete, Allomyces neo-moniliformis, was found to have a sedimentation coefficient of 2.3 S, and a molecular weight of approximately 30 000. The characteristics of this colchicine binding protein from Allomyces are compared to those of the 6 S colchicine binding proteins obtained from a variety of other organisms.