The addition of glucose-1-phosphate to the cytoplasmic glycoprotein phosphoglucomutase is modulated by intracellular calcium in PC12 cells and rat cortical synaptosomes.

In a number of different cell types, phosphorylation of a 63-kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus-sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester-linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [14C]glucose or the [beta-35S]phosphorothioate analogue of UDP-glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.

An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol.

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ([beta-35S]UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with [beta-35S]. UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue.

Denervated single myofibers: neurite interactions and synaptic molecules.

As a first step in defining the molecular cues that may be important for reinnervation of long-term denervated muscle, single adult rat muscle fibers that had been denervated from 2 to 24 months in vivo were maintained in culture for 5 days. Embryonic ventral spinal cord explants were added to some of these cultures. Interactions of neurites with individual short-term (up to 5 months) and long-term (17-24 months) denervated muscle fibers were compared with neurite interactions in cultures of young adult muscle fibers (from 3 to 5-month-old rats) or aged muscle fibers (from 17 to 26-month-old rats). We found the following. (1) Three molecules that are found at the neuromuscular junction (NMJ)--acetylcholinesterase (AChE), acetylcholine receptors (AChRs), and gelasmin (an acetylcholine receptor clustering factor that is found enriched at NMJ of adult muscle)--were reduced with increasing periods of denervation but not with aging. (2) The number of neurite contacts at junctional regions of muscle fibers that were formed and maintained on cultured muscle fibers depended on denervation time of the muscle in vivo; very few contacts were made or maintained on long-term denervated fibers. (3) Gelasmin, but not AChE or AChRs, was found at points of neurite contact on all muscle fibers examined, raising the possibility that it may serve as a cue for reinnervation and that its loss from long-term denervated muscle may be, at least in part, involved in the failure of neurite contacts to be made or maintained in culture and possibly in vivo.

Maintenance of neurite contacts at junctional regions of cultured individual muscle fibers from aged rats is correlated with the presence of a synapse-associated protein, gelasmin.

When adult skeletal muscle is denervated as a result of injury or disease, it can usually be reinnervated. Throughout the life of an animal, some skeletal muscles are thought to undergo cycles of denervation and reinnervation with concomitant remodelling of the neuromuscular junction, even in the absence of injury or disease. In old animals, this reinnervation process may be faulty and may not occur at all in some aged muscle fibers, leaving them permanently denervated. In general, the former endplate is the preferred site of reinnervation, which has led to the speculation that specific molecular cues persist, particularly in the basal lamina of this region, that may mediate endplate reinnervation. Although these molecular cues are as yet unidentified, one candidate is gelasmin, a 93 kD glycoprotein we have isolated and characterized from preparations of rat synaptic extracellular matrix. Because studies of reinnervation of aged muscle in vivo are extremely difficult to perform, we have devised a tissue culture model system of muscle reinnervation composed of isolated individual aged (17-26 months old) or young adult (3-5 months old; control) rat skeletal muscle fibers and embryonic (day 13 in utero) ventral spinal cord explants. We found that (1) gelasmin was present at all sites of nerve-muscle contact on muscle fibers from both young adult and aged animals over a 10-day culture period, that (2) twice as many aged (88%) as young adult fibers (41%) had neurite contacts in the former junctional region at 10 days and (3) gelasmin was found on significantly more aged (95%) than young adult fibers (60%) grown without nerve explants. Furthermore, although no extrajunctional contacts were found on young adult fibers by the end of the 10-day culture period, substantial numbers of extrajunctional contacts were seen on aged fibers; gelasmin was present at all of these contact sites. These results are consistent with the idea that gelasmin, which is made by muscle fibers, may act to mediate or stabilize the contacts made by reinnervating nerve and that aged muscle fibers may regulate gelasmin or similar molecules differently from young adult muscle fibers.

Localization of the glucose phosphotransferase to a cytoplasmically accessible site on intracellular membranes.

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in rat liver is a glycoprotein of 62 kDa. This acceptor was labeled in liver homogenates through incubation with the 35S-labeled phosphorothioate analogue of UDP-Glc, and its distribution following differential centrifugation was compared to that of the glycoproteins labeled by CMP-[3H]N-acetylneuraminic acid. Whereas 94% of the 3H-labeled macromolecules fractionated to the microsomal pellet, 85% of the 35S-labeled 62-kDa glycoprotein was found in the high-speed supernatant. The distribution of the Glc-phosphotransferase was also examined following differential centrifugation, and the bulk of the activity was found in the 100,000 x g pellet. In contrast to results obtained with the lumenal microsomal markers 4 beta-galactosyltransferase and mannose-6-phosphatase, however, optimal activity of the Glc-phosphotransferase was not dependent on the disruption of microsomal vesicles by detergent. In addition, Glc-phosphotransferase was degraded by exogenous proteases in the absence of detergent, whereas the lumenal markers were not. We conclude, therefore, that the 62-kDa acceptor glycoprotein is cytoplasmic and is glycosylated by the Glc-phosphotransferase at a site accessible to the cytoplasm. This may prove to be a model for the topography of glycosylation of other cytoplasmic glycoproteins as well.

Improved culture of individual muscle fibres with and without spinal cord explants in a collagen gel.

Suspension culture of single adult rat flexor digitorum brevis (FDB) muscle fibres in Vitrogen, a purified collagen, on tissue culture plastic or glass with mesh ring supports is superior to culture upon other substrates including collagen-, laminin-, or Vitrogen-coated tissue culture plastic. The Vitrogen gel-fibre mixture which attaches to glass or plastic provides at least 10 times more fibres per dish than does plating fibres on other substrates. Use of Vitrogen gel permits variable plating densities and the production of adequate numbers of cultures for long-term experimental comparisons of acetylcholinesterase (AChE) and rhodamine-alpha-bungarotoxin (RBTX) distribution on muscle fibres. Use of 40 micrograms/ml ovotransferrin (OT) instead of chick embryo extract in the culture medium significantly improves long-term survival. Cultured fibres, with or without the addition of ventral spinal cord explants. may also be examined with electrophysiological techniques.