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Uterine homogenates of cycling and early pregnant Sprague Dawley rats and purified rat urinary kallikrein showed similar curves of displacement of 125I-kallikrein binding to a polyclonal antibody. Uterine kallikrein concentration measured by RIA was 8.7 +/- 2 SEM ng/g wet weight during the cycle (n = 6 in diestrus and metestrus) and 20.8 +/- 2 SEM (n = 7) ng/g wet weight on Day 7 of pregnancy (P7) (p < 0.001). On P7, kallikrein concentration was increased 12.4-fold in the implantation nodes, as compared to the interimplantation segments. Uterine homogenates of rats on P7, submitted to DEAE-cellulose chromatography and Sephadex gel filtration, yielded two fractions containing kallikrein immunoreactivity and kininogenase activity, with molecular masses that ranged from 120-125 kDa and 39-43 kDa, respectively. In the RIA, both fractions displayed parallelism with purified kallikrein. Enzymatic activity was expressed after activation by trypsin. It was inhibited by aprotinin, PMSF, p-amino-benzamidine, and leupeptin, but not by soybean or ovomucoid trypsin inhibitors. Kallikrein mRNA was demonstrated by reverse transcriptase/polymerase chain reaction in uteri of nonpregnant and P7 rats. These results show that rat uterus synthesizes one or more serine proteases that are immunologically and enzymatically related to tissue kallikrein in the implantation node on P7--determined both by an increment of whole uterus kallikrein content and a depletion of the interimplantation segments--suggests that kallikrein may play a role in the vasoactive changes of implantation.

Externally added ascorbate or NADH effectively reduced ferricyanide and promoted the exit of Fe3+ originated from acid-destabilized transferrin contained inside endocytic vesicles. The effect of ascorbate was mediated by an ascorbate uptake system, and the effect of NADH was mediated by the membrane-associated oxidoreductase. At physiological concentrations of both ascorbate and NADH, the ascorbate transport and the NADH-oxidoreductase system were additive as measured by the rate of reduction of ferricyanide and by the mobilization of transferrin-associated iron. The results indicate that Fe3+ reduction may occur by a non-enzymatic reaction with ascorbate transported into the vesicle lumen. The ascorbate-mediated reduction of iron derived from transferrin occurring in the endosome could play a major role in cellular iron uptake.

The kinetics of the separate processes of Fe2(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and 59Fe, 125I-labeled rabbit transferrin. The binding of 59Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2 x 10(5) M-1 sec-1. The rate of internalization of 59Fe-transferrin was directly measured at 520 +/- 100 molecules of Fe2(III)-transferrin internalized/sec/cell with 250 +/- 43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe2(III)-transferrin internalization the flux of 59Fe was followed through three compartments: internalized transferrin, membrane, and cytosol. A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17 +/- 2 sec and 34 +/- 5 sec, respectively. Apparent rate constants of 0.0075 +/- 0.002 sec-1 and 0.0343 +/- 0.0118 sec-1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112 +/- 0.0025 sec-1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe2(III)-transferrin that are distinct from the handling of transferrin.

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