In mammalian cells two mannose 6‐phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation‐dependent mannose 6‐phosphate receptor (CD‐MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD‐MPR in vivo. However, homozygous receptor‐deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild‐types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD‐MPR is required for efficient intracellular targeting of multiple lysosomal enzymes.

Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in ritro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar N03-for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO3-likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO3-, the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO3-occurs during uptake and reduction of exogenous NO3-. Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO3-. It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO3-, (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO3-is taken up by cells of the cortex and stele, and/or (c) N03-is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO3-reduction.Experiments with wheat and corn seedlings using 15N03-have revealed that NO3-reduction in roots may be localized in a specific cellular compartment (1,17,18). Following induction of the N03 transport and reduction systems, exogenously supplied NO3-being taken up by the root was readily reduced and translocated to the xylem. In contrast, endogenous NO3-which had been previously accumulated in the tissue was readily translocated or effluxed to the external solution, but minimal amounts were reduced.

We have characterized a soluble form of the insulin-like growth factor II/mannose 6-phosphate receptor (sIGF-II/MPR) and bound ligands from bovine serum. Fetal serum contained 2-8 mg/liter sIGF-II/MPR. Affinity-purified receptor isolated by adsorption to phosphomannan-agarose and elution with mannose 6-phosphate contained nearly stoichiometric amounts of bound 7.5-kDa IGF-II. In addition, at least 12 distinct 12-20-kDa proteins immunologically related to IGF-II also copurified with receptor. Receptor was separated from its associated ligands by acidification and gel filtration chromatography. Sequence analysis revealed that the 12-20-kDa proteins have the same amino termini as mature 7.5-kDa IGF-II. Protease and glycosidase treatments revealed that the different high molecular weight IGF-II species contain an identical COOH-terminal extension that is differentially glycosylated with O-linked sugars. Radiolabeled tracer experiments demonstrated that the sIGF-II/MPR carries approximately 1/4 of the IGF-II in fetal bovine serum. These results support a significant role for sIGF-II/MPR in the transport of circulating IGF-II isoforms during development.

ABSTRACIThe appearance and disappearance of NADH:nitrate reductase (NR) in the leaves of corn (Zea mays L. W64A x W182E) were studied using activity assays, an enzyme-linked immunosorbent assay (ELISA) and western blotting. N-starved, etiolated corn plants were treated with nutrients containing either 35 millimolar NH4-nitrate or K-nitrate and immediately thereafter given light. The curve for enhancement of NR activity had three phases: I hour lag, 5 hour rapid increase, and steady state. The pattern for NR protein, as measured with the ELISA, also had three phases, but the increase was more rapid and the steady state was established earlier. To differentiate the effects of N nutrition from those of light, N-starved etiolated plants were given N nutrients 4 hours before light. During the dark pretreatment, NR activity and protein increased. When the light was turned on the NR activity and protein increased very rapidly without a lag. Western blots of polyacrylamide gels of native and denatured crude extracts showed that NADH:NR polypeptide was absent prior to treatment with N nutrients, but appeared after nitrate was given in dark or light. A low level of NR activity was found in N-starved, etiolated plants and it was shown by western blotting to be an NR form with a different electrophoretic mobility in nondenaturing gels. Since this minor NR form was not influenced by either nitrate or light, it was designated a constitutive NR. Dark decay of NR activity and protein was also studied. After the plants which had been in light with N nutrients for 24 hours were transferred to dark, the NR activity dropped by 30% within 1 hour, but the NR protein did not decrease. This inactivation of NR was further supported by returning the plants to the light after 1.5 hours of dark and finding the activity restored without change in NR protein. After the initial activity drop, a parallel decrease in NR activity and protein was observed, which was likely due to irreversible degradation by proteolysis.Hageman and Flesher (10) first studied NR2 activity in corn seedlings as affected by light and nitrate. They found rates of NR activity appearance greatly influenced by the previous N nutrition of plants when etiolated plants were greened by exposure to white light. The appearance of NR activity was co-dependent on light and nitrate in the 8-d-old, etiolated seedlings (10). From their study and others that have followed, a more or less standard response pattern for "induction" of NR activity by nitrate can

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