Affinity of phytoestrogens for estradiol-binding proteins and effect of coumestrol on growth of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors.

A number of nonsteroidal estrogens, which are common naturally occurring substances in human foods, were examined for competitive binding to estrogen receptor proteins. These compounds bound competitively to estrogen receptor proteins in rat uterine cytosol, in tissue from 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumors, and in human mammary tumor tissue. The relative affinity of these estrogens for rat uterine cytosol receptors paralleled closely the affinities reported for other receptors. Oral administration of coumestrol did not appear to support the growth of DMBA-induced rat mammary tumors, nor did coumestrol act as an antiestrogen when administered orally in combination with 17 beta-estradiol. Coumestrol administered sc might, however, be able to support the growth of these tumors.

Isolation and characterization of genetic variants of the Kunitz soybean trypsin inhibitor.

Three genetically related forms of the Kunitz soybean trypsin inhibitor have been isolated by ion-exchange chromatography. The three variants have different electrophoretic mobilities and have been designated Tia, Tib and Tic. An enzyme assay system was used to determine the association equilibrium constant of each variant for trypsin at pH 8.1 and values of 2 x 10(9) M-1, 2 x 10(7) M-1 and 3 x 10(10) M-1 were found for Tia, Tib and Tic respectively. The hydrolysis equillibrium constant at pH 6.0 for the reactive site peptide bond hydrolysis for each variant was also determined and found to be 4, 9 and 3 for Tia, Tib and Tic, respectively.

Kinetics of pepsin-initiated coagulation of kappa-casein.

The kinetics of pepsin initiated coagulation of kappa-casein have been studied at pH 5.8. The primary and secondary phases of coagulation are shown to proceed simultaneously. The theory of enzymatically initiated clotting reactions proposed by Payens (Payens, T.A.J. (1976) Neth. Milk Dairy J. 30, 55--59) has been applied to this clotting system and has been used to obtain rate constants for the secondary phase of coagulation. As expected, clotting rate constants for kappa-casein increase with pepsin concentration. An activation energy of 30.6 kcal/mol has been obtained for the secondary phase of coagulation. Turbidity measurements are a convenient means for studying the secondary phase of coagulation but do not provide an unambiguous means for studying the primary phase of the reaction.

Phytoestrogen interaction with estrogen receptors in human breast cancer cells.

The interactions of phytoestrogens with estrogen receptors were studied in the human breast cancer cell line, MCF-7. The compounds tested were coumestrol, genistein, and formononetin and the mycotoxins, zearalenone and its reduced derivative, zearalenol. All but formononetin compete for binding of [3H]-estradiol to unfilled cytoplasmic estrogen receptor or unfilled nuclear estrogen receptor sites. Relative binding affinities are zearalenol HMP (high melting point isomer) greater than zearalenol LMP (low melting point isomer) greater than zearalenone = coumestrol greater than genistein greater than formononetin. Dissociation constants estimated from competition curves show that binding affinities are high. In contrast to estradiol, phytoestrogens bind only weakly to sex steroid-binding globulin; they also do not bind to corticosteroid-binding globulin. These compounds translocate the cytoplasmic estrogen receptor and bind to unfilled nuclear estrogen receptors in whole cells. Bound nuclear receptors are then processed in a manner similar to estradiol in a step which rapidly decreases total cellular estrogen receptors. The phytoestrogens are also biologically active; they can markedly enhance tumor cell proliferation. In sum, phytoestrogens interact with the estrogen receptors of human breast cancer cells in culture and, therefore, may affect estrogen-mediated events in these cells.

Lack of mutagenicity of some phytoestrogens in the salmonella/mammalian microsome assay.

8 phytoestrogens were tested for mutagenicity using a variation of the Salmonella/mammalian microsome (or Ames) assay. Zearalenone is a mycotoxin produced by a grain contaminant, Fusarium graminearum (Gibberella zeae) and the isomers of zearalanol are reduced derivatives of this compound. The remaining compounds are all flavonoids which occur naturally at relatively high concentrations in many plants, particularly legumes. 4 of these flavonoids (daidzein, genistein, formononetin and biochanin-a) are isoflavones and the 5th, coumestrol, is a coumestan. Each compound was tested at several concentrations ranging from 1--500 micrograms per plate. The microsomal fracton was obtained from Aroclor 1254 (a PCB)-induced rat livers. None of the compounds tested was mutagenic to Salmonella strains TA1538, TA98 or TA100 at any concentration.

The interaction of inhibitors of proteolytic enzymes with 3-methylhistidine-57-chymotrypsin.

The inhibition of proteolytic enzymes by protein inhibitors is accompanied by the formation of unusually stable complexes. The recognition of a specific substrate-like amino acid on the inhibitor is believed to be the initial event of the inhibitory process. In addition to the interactions involved in the binding of a good substrate, a variety of other noncovalent interactions are known to stabilize the complex. The formation of stable complexes between several inactive derivatives of proteolytic enzymes and a variety of protein inhibitors suggests strongly that the formation of any species resembling catalytic intermediates is unnecessary for inhibition. We have examined the interaction between several avian ovomucoids and alpha-chymotrypsin in which histidine-57 has been converted to 3-methylhistidine-57. This derivative is easily prepared and can be isolated by affinity chromatography. Methylchymotrypsin retains unaltered its ability to bind specific substrates, but is essentially inactive. In spite of this loss of enzymatic activity, methylchymotrypsin forms strong complexes with several inhibitors. In addition, methylchymotrypsin which has been covalently linked to Sepharose is particularly useful for the isolation of protein inhibitors without the complications due to isolation of a mixture of partially cleaved forms of the inhibitor.

Affinity Chromatography of the Major Seed Protein of the Bean (Phaseolus vulgaris L.).

The major globulin of the French bean (Phaseolus vulgaris L.) undergoes a reversible pH-dependent polymerization. At pH values above 6.5, the monomeric form of the protein predominates; and at pH values below 6.5, the protein occurs as a polymer, probably a tetramer. At extremes of pH, the protein dissociates further into peptides. The reversible pH-dependent interaction between globulin subunits is used in this report as the basis for an affinity chromatography procedure for isolation of the globulin. The major globulin from several genetic variants can be obtained in gram quantities and does not indicate the presence of any impurities on discontinuous sodium dodecyl sulfate gel electrophoresis.