Lysine reduces renal accumulation of radioactivity associated with injection of the [177Lu]alpha-[2-(4-aminophenyl) ethyl]-1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic acid-CC49 Fab radioimmunoconjugate.

The high uptake and prolonged renal retention of monoclonal antibody fragments that are conjugated with radiometal chelates precludes their routine clinical use due to high background counts, which may hinder detection of nearby lesions and/or cause renal radiotoxicity. We report on the potential use of Lys as a pharmacological agent to enhance renal excretion of the [177Lu]alpha-[2-(4-aminophenyl) ethyl]-1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic acid CC49 Fab ([177Lu]CC49 Fab) radioimmunoconjugate. The monoclonal antibody portion of this complex is directed toward the tumor-associated glycoprotein-72 antigen. Lys was administered to female BALB/c mice by i.p. injections. [177Lu]CC49 Fab bolus injections were given by the i.v. route. Results of our investigations showed that: (a) kidney radioactivity concentrations were inversely related to Lys dose. The optimal dose (50 mg/mouse) evoked a 3-fold reduction in kidney counts; (b) Lys was most effective when injected 15 min before, or at the same time as, [177Lu]CC49 Fab; (c) the renal effect was both rapid (3-fold decrease at 15 min after injection) and prolonged (4-fold decrease at 24 h after injection); (d) a single Lys dose decreased total body radioactivity by > 2.5-fold; (e) urine excretion of radioactivity was enhanced in Lys-treated mice. High pressure liquid chromatographic analyses using a GF-250 column showed that a large fraction of this urine radioactivity coeluted with a [177Lu]CC49 Fab injection standard. We conclude that Lys enhances the urinary excretion of radioactivity associated with [177Lu]CC49 Fab. These observations warrant further study with regard to the use of amino acids or their derivatives as pharmacological agents to enhance the urinary excretion of small-molecule radioimmunoconjugates.

Comparison of rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin--II. Immunological properties.

Purified rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin have been immunologically compared. A comparison using the steady state gel method of Ritzen et al. indicated immunological cross-reactivity. In order to further compare their immunological properties we developed a radioimmunoassay for both rbABP and rbTeBG using specific antisera directed against each. When these assays were compared, the extent or completeness of displacement proved to be the only parameter that was significantly different. This data obtained with homologous and heterologous radioimmunoassays is consistent with the idea that these two proteins contain minor antigenic determinants which are distinct.

Specific adsorption of a platelet membrane glycoprotein by human insoluble collagen.

A platelet membrane glycoprotein, 61 kDa, has been identified, which binds specifically to insoluble collagen. The detection of this protein was accomplished by incubating radiolabeled Triton-solubilized platelet supernatant with insoluble collagen, and, after washing the collagen pellet, extracting the 61-kDa glycoprotein from the pellet with sodium dodecyl sulfate buffer. The optimal conditions for specific binding were incubation of 120 micrograms of total platelet supernatant protein with 2 mg of collagen at 4 degrees C for 0.5 h in 0.5 ml of the incubating buffer (20 mM Tris, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, and 0.2% Triton, pH 7.4). The 61-kDa glycoprotein is cleaved by trypsin into a major peptide (44 kDa) and a smaller peptide(s) linked together by disulfide bonds in a molecule which still binds to collagen. When intact platelets are treated first with trypsin and then with dithiothreitol, the 44-kDa peptide is released and was shown to bind to collagen. We conclude that the 61-kDa glycoprotein is a platelet membrane protein which specifically interacts through its extracellular domain with insoluble collagen, and, thus, must be considered as a possible component of the initial platelet-matrix adhesion process which leads to platelet aggregation in vivo.

Interaction of specific platelet membrane proteins with collagen: evidence from chemical cross-linking.

Two recently developed membrane-impermeant cross-linkers, 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) and bis(sulfosuccinimidyl) suberate (BS3), have been used to examine the interaction of human platelets with collagen. Reaction of human platelets with either of the two cross-linking reagents at micromolar concentrations completely inhibited platelet aggregation in response to collagen but not in response to thrombin. Platelet adhesion to collagen was, however, not affected by these reagents. Inhibition of collagen-induced platelet aggregation by DTSSP or BS3 appears to be due to cross-linking and not simply to the chemical modification of membrane proteins, since the homologous monofunctional reagent sulfosuccinimidyl propionate had no effect on platelet aggregation. Inhibition of platelet aggregation by BS3 was accompanied by a decrease in the intensity of glycoprotein bands IIb, IIIa, and IV when analyzed on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels. In order to determine if collagen is directly interacting with a specific platelet membrane glycoprotein, 3H-labeled platelets were allowed to adhere to collagen and then cross-linked with various concentrations of DTSSP. Proteins which remain associated with collagen after lysis and washing were analyzed on NaDodSO4 gels. At concentrations of 16-50 microM DTSSP, glycoproteins IIb and IIIa appeared to be specifically cross-linked to collagen. These results suggest that the glycoprotein IIb-IIIa complex, which has previously been implicated as the fibrinogen receptor in activated platelets, may also be directly involved in collagen-induced platelet aggregation.

Subunit structure of rabbit testosterone estradiol-binding globulin.

Testosterone estradiol-binding globulin (rbTeBG) of rabbit plasma was purified to homogeneity by the sequential use of ammonium sulfate precipitation, affinity chromatography on 5 alpha-dihydrotestosterone-Sepharose, reactive blue-Sepharose, and finally preparative polyacrylamide gel electrophoresis. An overall purification of greater than 3000-fold was achieved with a recovery of 32%. This purified material was shown to possess the following physical characteristics: an equilibrium dissociation constant at 4 degrees C for dihydrotestosterone estimated to be 0.59 X 10(-9) M and a half-time of dissociation of rbTeBG-dihydrotestosterone complex of approximately 9 min at 4 degrees C. The purified protein shows considerable microheterogeneity with regard to size on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and to charge on isoelectric focusing gels. Analytical gel electrophoresis in the presence of SDS revealed two molecular weight components of 41,000 and 45,000. Size analysis on native gels and cross-linking studies yields a molecular weight of 101,600 and 83,000, respectively. These size components were shown to be very closely related with regard to primary structure. rbTeBG was completely absorbed by concanavalin A Sepharose resin, demonstrating the glycoprotein nature of rbTeBG. Highly purified rbTeBG and unpurified rbTeBG from rabbit plasma could be photolabeled with [3H]delta 6-testosterone and when analyzed on SDS-polyacrylamide gel electrophoresis (PAGE), two major bands of radioactivity with molecular weights of 41,000 and 45,000 appeared in both samples.

Is FSH required for maintenance of spermatogenesis in adult rats?

Administration of FSH antiserum to adult rats for 14 or 30 days had no or little effect on body, testis or accessory sex gland weights, androgen-binding protein, testosterone levels, germ cell numbers or fertility, thus indicating a relative insensitivity of the testis to withdrawal of FSH. Unlike immature rats, therefore, which do require FSH to initiate spermatogenesis, adult rats do not need this hormone to maintain spermatogenesis.