The in vitro bioactivity of the human beta TSH subunit was investigated utilizing eleven overlapping synthetic peptides representing the entire 112 residue sequence. The peptides were tested for both stimulatory and inhibitory activity in two sensitive bioassay systems: the first based on cAMP production in FRTL-5 rat thyroid cells, and the second based on stimulation of iodine trapping by the same continuous cell line. Peptides from three distinct regions of the beta-subunit showed concentration dependent inhibition of TSH bio-activity, including beta 1-15, beta 11-25, beta 31-45, beta 81-95, and beta 91-105 with IC50 values ranging from 150 to 304 microM. An additional peptide representing the entire sequence of the "intercysteine loop" region of beta TSH, beta 31-52, also inhibited TSH activity with somewhat higher potency than its fragment peptide beta 31-45 (IC50 of 87.5 +/- 14.7 microM for beta 31-52 versus 207 +/- 92.4 microM for beta 31-45). Three of these, beta 1-15, beta 31-45, and beta 31-52, also inhibited binding of TSH to the receptor in a radio-receptor assay, as previously reported (1), supporting their importance in receptor interaction. None of the synthetic peptides stimulated either cAMP production or iodine trapping. Two other overlapping peptides, beta 81-95 and beta 91-105, possessed bio-inhibitory activity but did not inhibit binding of labeled TSH. Computer analysis of this sequence predicted an extended turn structure for this region. This region has been referred to as the "determinant loop" as it is bounded by cysteine residues at positions 88 and 95 that many believe form a disulfide bond in the native subunit. The current data suggests the beta 88-95 region may play a role in receptor activation after initial binding of hormone to receptor.

Membrane fractions from fully luteinized rat ovaries contained proteolytic enzymes that were solubilized and reversibly inhibited by the anionic detergent sodium dodecyl sulfate. Electrophoretic analysis in substrate-containing sodium dodecyl sulfate-polyacrylamide slab gels revealed the presence of numerous protease bands in the mol wt range of 26,000 to more than 200,000. When casein served as protease substrate in the gels, enzyme bands of Mr 26,000, 28,000, 30,000, and 90,000 were produced by crude (2,000 X g pellet) ovarian membranes. Gels containing casein and plasminogen also revealed the presence of two plasminogen activators of Mr 63,000-65,000 and 42,000. Subcellular fractionation of luteinized ovaries by centrifugation in sucrose density gradients indicated that the Mr 90,000 protease and the two plasminogen activators were uniformly distributed among microvillous membranes, basolateral membranes (BLM), and the mitochondrial-lysosomal fraction (MLF). The Mr 26,000, 28,000, and 30,000 proteases were enriched in BLM and MLF. Analysis of crude membranes in slab gels which contained gelatin as the protease substrate revealed the presence of two additional enzymes of Mr 52,000 and more than 200,000. The Mr greater than 200,000 protease was present in microvillous membranes, BLM, and MLF. The Mr 52,000 protease was found exclusively in BLM. This enzyme was not consistently demonstrable in crude membranes but could be generated upon incubation of membranes at 30 C. This finding indicated that Mr 52,000 protease can exist in an inactive and/or zymogen form. The Mr 90,000 protease was inhibited by tosyl-lysine chloromethyl ketone and dansyl-glutamyl-glycylarginine chloromethyl ketone. The gelatinase activity of the Mr 52,000 protease was blocked by tosyl lysine chloromethyl ketone, dansyl-glutamyl-glycylarginine chloromethyl ketone and tosylamide-2-phenyl chloromethyl ketone. The activity of the Mr 26,000, 28,000, and 30,000 proteases was not affected by any of the above mentioned inhibitors. These findings demonstrate that the proteolytic potential of ovarian membranes is not limited to plasminogen activators. Numerous plasminogen-independent proteases are also present, and these may play a role in ovulation, luteolysis, and mediation of hormonal stimulation.

Filtration through polyethylenimine (PEI)-treated membrane filters efficiently separates free radiolabeled human (h) LH or hCG from solubilized receptor-hormone complexes. Solubilized LH/hCG receptors of bovine, porcine, and rat origin are suitable for measurement by this method. Specific binding assayed by the PEI-filter technique was 106-138% of specific binding assayed by double polyethylene glycol precipitation, demonstrating that the method gave quantitative recovery of bound hormone. Nonspecific binding was consistently lower with PEI-treated filters. Intra- and interassay variability for the two different methods was similar. These results indicate that filtration through PEI-treated membrane filters is a simple and rapid method for assay of solubilized LH/hCG receptors.

Tumor cell killing effect of hematoporphyrin derivative (HPD) and light was studied in culture to determine the dependence of this effect on treatment variables. Particular attention has been given to the spectral characteristics of the light and the absorption properties of hematoporphyrin. A human tumor cell line was treated using HPD and three broad bands of light ranging from the short- to the long-wavelength end of the visible spectrum. Cell killing was assessed by trypan blue exclusion. A transformed mouse embryo cell line was treated in a similar manner, and its reproductive efficiency was determined following treatment. Results of both studies are consistent with the hypothesis that the photocytotoxic action of HPD plus light is directly proportional to the number of light quanta absorbed by the HPD in each cell. For thin layers of cells, such as in situ carcinoma, it appears that short-wavelength radiation falling in the porphyrin Soret band around 400 nm may have from 12 to 30 times the killing power as does red light.

The effects of a number of proteinase inhibitors on rat ovarian and rat hepatic adenylate cyclase preparations were examined. N alpha-tosylarginine methyl ester, 7-amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one, 1-chloro-4-methyl-3-L-tosylamidopentan-2-one and other low-molecular-weight proteinase inhibitors blocked hormonally stimulated adenylate cyclase from either source with hepatic preparations requiring higher concentrations. Addition of nucleotides (ATP, GTP, GDP, CTP or ITP) to inhibited ovarian preparations did not reverse inhibition, nor did dithiothreitol reverse phenylmethanesulphonyl fluoride-inhibited ovarian adenylate cyclase. The kinetics of the inhibition of rat ovarian adenylate cyclase were examined by following the production of cyclic AMP after the addition of inhibitors to membrane preparations preincubated under assay conditions with human choriogonadotropin, guanosine 5'-[beta gamma-imido]triphosphate of NaF. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one and 1-chloro-4-methyl-3-L-tosylamidopentan-2-one had two effects on human-choriogonadotropin-stimulated adenylate cyclase. At low concentrations (less than or equal to 0.2 mM) there was an irreversible inhibition of hormonally-stimulated cyclase with maximum first-order inhibitory rate constants of 0.05--0.08 min-1. At higher concentrations the irreversible effect persisted, but, in addition, there was a marked decrease in the cyclase initial velocity to 25--50% of that of control values. N alpha-tosylarginine methyl ester had similar effects; at low concentrations (less than or equal to 2 mM) it inhibited irreversibly, and at higher concentrations it decreased the initial velocity (50% at 10 mM). At high concentrations (greater than 3 mM) N alpha-tosylarginine methyl ester also inhibited NaF- and guanosine 5'-[beta gamma-imidol]-triphosphate-stimulated cyclase but in a reversible manner. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one inhibited NaF-stimulated adenylate cyclase in two ways, as for human-choriogonadotropin-stimulated adenylate cyclase, but required 10--20-fold higher concentrations. The low-concentration irreversible effect can be explained by a continual inactive in equilibrium active conversion of adenylate cyclase during hormonal stimulation in which the inactive to active conversion is blocked by the inhibitors. The high-concentration effect is a direct one on the active catalytic moiety of the enzyme.

Previous studies have shown that human chorionic gonadotropin stimulation of rat ovarian adenylate cyclase activity is enhanced but not augmented by GTP. Extraction of crude rat luteal membranes with 2M urea induced a GTP augmentation of hCG stimulation of adenylate cyclase activity of 3.5 fold. This augmentation of hormonal stimulation of adenylate cyclase activity by GTP was not accompanied by a decrease in hormone binding such as has been observed with other hormonally stimulated adenylate cyclase.

Porcine granulosa cells from small follicles were incubated in the presence of 1 muCi/ml 3H-glucosamine and various hormones. Proteoglycans were digested with protease, and the mucopolysaccharides (MPS) were recovered. Most of the labeled MPS was found in secretory products. Maximum incorporation of 3H-glucosamine into MPS occurred after incubation with FSH for 8--16 h. Cell concentrations of 0.5--4.0 x 10(6)/ml proved to be ample. Highly purified porcine or human FSH gave consistent, significant stimulation by doses in the order of 100 pg/ml. Linear log-dose response curves occurred from 0.1 to 10.0 ng/ml, and maximal stimulation at 10 ng/ml was 22-fold above control. Preparations of hCG, hTSH and NIH-LH-S18 were not effective. Incubations with 1--10 mM dibutyryl cyclic AMP significantly increased incorporation into MPS, suggesting that effects of FSH may be mediated by cyclic AMP.

We recently published evidence of the presence of circulating antibodies in the sera of patients who had the diagnosis of premature menopause syndrome. This led to the question: Is there a circulating antibody in the sera of patients with premature menopause syndrome which interferes with gonadotropin-luteinizing hormone (LH) receptor interaction on the luteal cell surface? Serum samples from 14 women with the diagnosis of Premature menopause syndrome were examined to see whether they would block the binding of 125I-labeled human chorionic gonadotropin (hCG) to human corpora lutea. All patients had spontaneous cessation of menses before the age of 35 years and documentation of increased gonadotropin secretion and failure of estrogen secretion. When the sera of the study group were incubated with 125I-labeled hCG and LH receptor from human corpora lutea, no difference in binding was observed when compared with controls. This suggests that there is no circulating antibody that interferes with hCG-LH receptor interaction and that would thereby lead to loss of ovarian function in premature menopause syndrome.

Structural studies have substantiated the concept that the glycoprotein hormones consist of a "common" alpha subunit and "hormone-specific" beta subunit. Despite this, consensus is still lacking concerning certain portions of the amino acid sequences, including alignment of residues 81--82 in the alpha subunit. We have carried out sequence analysis of the alpha subunit of human luteinizing hormone (hLH) to clarify the assignment of these residues and to examine further the nature of the amino-terminal heterogeneity found among the different alpha subunits. Our structure showed residues 80--84 to be -His-Cys-Ser-Thy-Cys-, consistent with findings of others for human follicle stimulating hormone (hFSH) and human chorionic gonadotropin (hCG). The extensive degree of heterogeneity found in the amino terminal region of hFSH and hCG is present to only a minor extent in hLH. The differences in the pattern of amino-terminal heterogeneity among the various hormones may result from differences in the nature of cleavage of subunit from a larger intracellular precursor peptide.

Hematoporphyrin derivative (HpD) is one of several photodynamically active dyes that accumulate in tumor cells and can be used to differentiate neoplastic from normal tissue by violet light activation. Degree of tumor fluorescence was studied in relation to the nature of the hematoporphyrin compound, dose of HpD injected, route of injection, and intensity of activating light. Our studies indicate that HpD is not a chromatographically homogeneous material. Four components of HpD have been identified by thin-layer chromatography; their nature is not presently known. Pure HpD is the best agent for tumor fluorescence as compared with the components studied. Intravenous injection of HpD resulted in a more intense red-orange fluorescence than that after intraperitoneal injection. Fluorescence was more intense at the tumor periphery where tumor invaded normal tissues and around blood vessels. HpD injection of 0.002 mg/gm body weight induced more selective fluorescence of tumor than did 0.01 mg/gm. Microscopic examination of tumor showed fluorescence distributed throughout the cytoplasm. A fiberoptic bronchoscope attached to the Mayo-Baldes activating light source made possible induction of slight tumor fluorescence, but the intensity of activating light would not be adequate for clinical examination of the bronchial tree and detection of tumor.

The normal midcycle surge of luteinizing hormone (LH), an approximately 10-fold increase, and a smaller surge of follicle-stimulating hormone (FSH) are preceded by peaks in serum estradiol (E2) and 17alpha-hydroxyprogesterone (17-P). The elevation in E2 is thought to be the important trigger mechanism for the LH surge. The possibility that the preovulatory elevation in 17-P may by itself, or combined with E2, be the trigger mechanism was investigated. E2 ans 17-P, alone and combined, were infused to mimic late follicular-phase blood concentrations in postmenopausal women pretreated with E2. This produced 1.6- to 4.6-fold increases in serum LH and lesser increases in serum FSH 24 hours after infusion. Thus, the normal ovulatory surge of gonadotropins could not be reproduced by infusions of 17-P or E2, or both. Infusion with 17-P appeared to stimulate LH release and did not augment or inhibit the effect of E2 on gonadotropin release.

Ovarian receptors specific for hCG-LH can be induced by PMSG-ovine LH priming. The marked increase in binding activity was accompanied by a rise in ovarian progesterone concentration and preceded by ovarian weight increase. The high binding activity of heavily luteinized ovaries was followed by a decline to prepriming levels during luteolysis. The ovarian progesterone content was decreased simultaneously and no change in ovarian weight was evident. The data demonstrate that ovarian hCG binding activity undergoes variation which depends on the functional state of ovaries.