Studies on the microheterogeneity and in vitro activity of glycosylated and nonglycosylated recombinant human prolactin separated using a novel purification process.

Recombinant human PRL was produced in a murine C127 cell expression system and purified to greater than 97% homogeneity using anion and cation exchange chromatography. This material was biologically equivalent to pituitary-derived PRL in both an enzyme-linked immunosorbent assay and the Nb2 lymphoma cell proliferation assay. The predominant PRL forms were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as being 23 and 25 kilodaltons (kDa). These mass values were confirmed by electrospray mass spectroscopy. Glycosidase digestions indicated that the 25-kDa PRL is N-glycosylated and sialylated, whereas 23-kDa PRL is nonglycosylated. Glycosylated and nonglycosylated forms of the hormone were individually purified to greater than 95% homogeneity using novel cation exchange chromatography. Isoelectric focusing demonstrated that both forms consist of multiple charge isomers, with the charge heterogeneity of the glycosylated form primarily due to differences in sialylation. Monosaccharide analysis of the glycosylated form suggested a minimal complex oligosaccharide chain that may be fucosylated and partially sialylated. Oligosaccharide mol wt were determined by electrospray ionization mass spectroscopy. Analysis of the oligosaccharides by fluorophore-assisted carbohydrate electrophoresis indicated that bi- and triantennary oligosaccharide forms are predominant and have multiple combinations of terminal sialylation. Both forms of PRL were active in the Nb2 lymphoma cell proliferation assay; however, the 23-kDa nonglycosylated form was 3-4 times more active in this assay than the 25-kDa glycosylated form.

Aluminum inhibits calpain-mediated proteolysis and induces human neurofilament proteins to form protease-resistant high molecular weight complexes.

We studied the effects of aluminum salts on the degradation of human neurofilament subunits (NF-H, NF-M, and NF-L, the high, middle, and low molecular weight subunits, respectively) and other cytoskeletal proteins using calcium-activated neutral proteinase (calpain) purified from human brain. Calpain-mediated proteolysis of NF-L, tubulin, and glial fibrillary acidic protein (GFAP), three substrates that displayed constant digestion rates in vitro, was inhibited by AlCl3 (IC50 = 200 microM) and by aluminum lactate (IC50 = 400 microM). Aluminum salts inhibited proteolysis principally by affecting the substrates directly. After exposure to 400 microM aluminum lactate and removal of unbound aluminum, human cytoskeletal proteins were degraded two- to threefold more slowly by calpain. When cytoskeleton preparations were exposed to aluminum salt concentrations of 100 microM or higher, proportions of NF-M and NF-H formed urea-insoluble complexes of high apparent molecular mass, which were also resistant to proteolysis by calpain. Complexes of tubulin and of GFAP were not observed under the same conditions. Aluminum salts irreversibly inactivated calpain but only at high aluminum concentrations (IC50 = 1.2 and 2.1 mM for aluminum lactate and AlCl3, respectively), although longer exposure to the ion reduced by twofold the levels required for protease inhibition. These interactions of aluminum with neurofilament proteins and the effects on proteolysis suggest possible mechanisms for the impaired axoplasmic transport of neurofilaments and their accumulation in neuronal perikarya after aluminum administration in vivo.

Multiple fates of newly synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons.

We have studied the fate of neurofilament proteins (NFPs) in mouse retinal ganglion cell (RGC) neurons from 1 to 180 d after synthesis and examined the proximal-to-distal distribution of the newly synthesized 70-, 140-, and 200-kD subunits along RGC axons relative to the distribution of neurofilaments. Improved methodology for intravitreal delivery of [3H]proline enabled us to quantitate changes in the accumulation and subsequent decline of radiolabeled NFP subunits at various postinjection intervals and, for the first time, to estimate the steady state levels of NFPs in different pools within axons. Two pools of newly synthesized triplet NFPs were distinguished based on their kinetics of disappearance from a 9-mm "axonal window" comprising the optic nerve and tract and their temporal-spatial distribution pattern along axons. The first pool disappeared exponentially between 17 and 45 d after injection with a half-life of 20 d. Its radiolabeled wavefront advanced along axons at 0.5-0.7 mm/d before reaching the distal end of the axonal window at 17 d, indicating that this loss represented the exit of neurofilament proteins composing the slowest phase of axoplasmic transport (SCa or group V) from axons. About 32% of the total pool of radiolabeled neurofilament proteins, however, remained in axons after 45 d and disappeared exponentially at a much slower rate (t 1/2 = 55 d). This second NFP pool assumed a nonuniform distribution along axons that was characterized proximally to distally by a 2.5-fold gradient of increasing radioactivity. This distribution pattern did not change between 45 and 180 d indicating that neurofilament proteins in the second pool constitute a relatively stationary structure in axons. Based on the relative radioactivities and residence time (or turnover) of each neurofilament pool in axons, we estimate that, in the steady state, more neurofilament proteins in mouse RGC axons may be stationary than are undergoing continuous slow axoplasmic transport. This conclusion was supported by biochemical analyses of total NFP content and by electron microscopic morphometric studies of neurofilament distribution along RGC axons. The 70-, 140-, and 200-kD subunits displayed a 2.5-fold proximal to distal gradient of increasing content along RGC axons. Neurofilaments were more numerous at distal axonal levels, paralleling the increased content of NFP.(ABSTRACT TRUNCATED AT 400 WORDS)