Heterogeneity of Soluble Neural Cell Adhesion Molecule

Soluble neural cell adhesion molecule (NCAM) from rat brain neuronal cell culture media consists predominantly of a polypeptide of Mr approximately 115,000. Minor amounts of a polypeptide of Mr approximately 180,000 and two inconsistently appearing components of Mr 160,000 and 145,000 are also observed. The Mr 115,000 component is derived from the neuronal membrane NCAM components NCAM-A of Mr 190,000, NCAM-B of Mr 140,000, or both. Thus, as a part of the catabolism of membrane NCAM-A plus -B, a minor fraction is posttranslationally cleaved and recovered in the media as discernible soluble NCAM polypeptides. The half-life of membrane NCAM-A plus -B is less than 24 h. Astrocyte culture media contains a predominant soluble NCAM component of Mr 120,000 derived from membrane-associated NCAM-C. A close comparison of deglycosylated soluble NCAM from astrocyte and neuronal cultures showed a small but consistent difference in Mr, a result suggesting that different NCAM polypeptides are released from the membrane of neurons and astrocytes. In contrast to the Mr 115,000-120,000 NCAM polypeptides, the Mr 180,000 polypeptide from neuronal culture media does not seem to be derived from membrane-attached NCAM and may therefore represent a secreted NCAM isoform.     

Solubilization of the omega-conotoxin receptor associated with voltage-sensitive calcium channels from bovine brain

The omega-conotoxin receptor in brain membranes contains components of Mr approximately equal to 310,000, approximately equal to 230,000, and 37,000 as identified by photoaffinity labeling. The toxin specifically bound to two sites with apparent dissociation constants (Kd) of approximately 3 pM and 3.5 nM under the conditions employed. There was about 8 times more of the low affinity site than the high affinity site. Binding was not affected by dihydropyridines or verapamil. However, diltiazem stereospecifically inhibited the binding to the high affinity site. Dissociation of the toxin from the membranes was very slow and only partial. Among the detergents tested, digitonin solubilized the highest toxin-binding activity. The digitonin extract contained only a single class of binding sites with an apparent Kd of about 0.46 nM. Probably only the high affinity binding site was recovered in active form in digitonin extract. The properties of the toxin binding to digitonin extract were in good agreement with those of the binding to the high affinity site in the original membranes. Photoaffinity labeling of the digitonin extract indicated that the solubilized toxin receptor contained the two large components (Mr congruent 310,000 and approximately equal to 230,000) observed in the membranes.     

Mechanism of biosynthesis of soluble and membrane-bound forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells

Dopamine beta-hydroxylase was present as 2 subunit forms (apparent Mr = 77,000 and 73,000) in the PC12 pheochromocytoma cell line as detected by immunoprecipitation from [35S]methionine-labeled cultures, and analyzed by sodium dodecyl sulfate gel electrophoresis and fluorography. The Mr = 77,000 form was present in a crude membrane fraction, while the Mr = 73,000 form was soluble. Both forms appeared to be present in approximately equal amounts, and both were glycosylated. Treatment of PC12 cells with tunicamycin, a potent inhibitor of core glycosylation in the endoplasmic reticulum, completely inhibited the appearance of the Mr = 77,000 and Mr = 73,000 forms, and 2 new immunoreactive polypeptides were obtained (apparent Mr = 67,000 and 63,000). Pulse-chase experiments suggested that the Mr = 77,000 form is initially synthesized (by 5 min) and a portion is converted in 15-90 min to the Mr = 73,000 form. Thereafter, the ratio between forms remains relatively constant, at least for several hours. Translation of mRNA from bovine and rat adrenals, and immunoprecipitation, indicated that dopamine beta-hydroxylase is initially synthesized as a single polypeptide (apparent Mr = 67,000). The subcellular site of biosynthesis of dopamine beta-hydroxylase was determined by isolation of mRNA from free and membrane-bound polysomes from bovine adrenal medulla. Translation in a cell free system and immunoprecipitation localized the synthesis of dopamine beta-hydroxylase on membrane-bound polysomes. These experiments suggest that both soluble and membrane-bound forms of dopamine beta-hydroxylase are synthesized and core glycosylated in the endoplasmic reticulum, and that there probably is a precursor-product relationship between the Mr = 77,000 and the Mr = 73,000 subunit forms of dopamine beta-hydroxylase.     

Nerve growth factor enhances expression of neuron-glia cell adhesion molecule in PC12 cells

The neuron-glia cell adhesion molecule (Ng-CAM) has been identified in mammalian brain tissue and PC12 pheochromocytoma cells as Mr 200,000 and Mr 230,000 species, respectively. When PC12 cells were treated with nerve growth factor (NGF), the amount of Ng-CAM at the cell surface was increased approximately threefold, whereas the amount of the neural cell adhesion molecule (N-CAM) remained unchanged. An NGF-inducible large external glycoprotein (NILE) has been previously identified by its enhanced expression in NGF-treated PC12 cells. Ng-CAM and NILE are similar in molecular weight, expression during development, and responsiveness to NGF in PC12 cells, suggesting that the two molecules are related. In addition, antibodies to Ng-CAM and NILE cross-reacted and the molecules had similar peptide maps after limited proteolysis. Moreover, antibodies to Ng-CAM inhibited fasciculation of neurites, a functional property shared with NILE. The results show that cell adhesion molecules can respond selectively to growth factors and suggest that NILE is, in fact, mammalian Ng-CAM.     

Endoglycosidase H susceptibility of secreted and intracellular forms of dopamine beta-hydroxylase

In an attempt to characterize the differences between various forms of dopamine beta-hydroxylase (DBH) the endoglycosidase H (endo H) susceptibilities of the intracellular and secreted DBH were compared. Both soluble and membrane-bound forms of newly synthesized DBH in PC12 cells were found to be susceptible to endo H. Soluble DBH, apparent Mr of 73K, was converted to 67K Mr and membrane-bound DBH, apparent Mr of 77K and 73K, shifted to 69K and 67K. Upon stimulation with the potent secretagogue, BaCl2, over 65% of intracellular dopamine and norepinephrine were secreted together with the soluble proteins of the secretory granules. DBH, released upon this stimulated secretion, was relatively resistant to endo H compared to the intracellular forms.     

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1.

An exo-beta-1,4-glucanase (Exo A) from Ruminococcus flavefaciens FD-1 was purified to homogeneity and characterized. Enzyme activity was monitored during purification by using the substrate p-nitrophenyl-beta-D-cellobioside (NPC). Over 85% of the NPC activity was found to be extracellular once the filter paper was degraded (7 days). Culture supernatant was harvested, and the protein was concentrated by ultrafiltration. The retentate (greater than or equal to 300,000 Mr), containing most of the activity against NPC, was then fractionated with a TSK DEAE-5PW column. This yielded a sharp major peak of NPC enzyme activity, followed by a broader, less active area that appeared to contain at least six minor peaks of lower enzymatic activity. Further purification was achieved by chromatography with a hydroxylapatite column. Finally, gel filtration chromatography yielded a homogeneous enzyme (Exo A) as determined by silver stains of both sodium dodecyl sulfate- and nondenaturing electrophoresis gels. Substrate specificity experiments and the products of cellulose digestion indicate that the enzyme was an exo-beta-1,4-glucanase. Exo A required Ca2+ for maximal activity and had an apparent Km of 3.08 mM for NPC, with a Vmax of 0.298 mumol/min per mg of protein. The enzyme had an Mr of 230,000, as determined by gel filtration chromatography, and was a dimer of 118,000-Mr subunits. The N-terminal amino acid sequence of the enzyme is presented.     

Characterization of a neuronal subtype of insulin-like growth factor I receptor

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.     

Structural Features of the Nerve Growth Factor Inducible Large External Glycoprotein of PC12 Pheochromocytoma Cells and Brain

Abstract: We have examined the oligosaccharide structure of a major M_r= 230,000 cell surface glycoprotein from rat PC12 pheochromocytoma cells, and of the immunochemically cross-reactive species present in brain. In response to nerve growth factor (NGF) the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. These morphological changes are accompanied by a 3- to 5-fold increase in the concentration and labeling of this cell surface glycoprotein, which has previously been named the NGF-inducible large external, or NILE, glycoprotein. Tri- and tetraantennary complex oligosaccharides are the predominant carbohydrate units present in the NILE glycoprotein from both brain and PC12 cells, where they represent 77–90% of the biosynthetically labeled oligosaccharides. Most of these are not substituted by fucose on the core N -acetylglucosamine which is linked to asparagine, and are accompanied by smaller proportions of biantennary and high-mannose oligosaccharides. Sequential lectin-agarose affinity chromatography employing concanavalin A, lentil lectin, and the leukoagglutinating lectin of Phaseolus vulgaris , together with neuraminidase treatment of the fractionated glycopeptides, demonstrated a moderate degree of microheterogeneity among the predominant tri- and tetraantennary oligosaccharide units with respect to the presence of core fucose, outer galactose and sialic acid residues, and the substitution positions on the α-linked mannose residues. NGF treatment of the PC12 cells had no significant effect on the oligosaccharide structure of the NILE glycoprotein. The greater molecular size of the PC12 cell NILE glycoprotein as compared to the immunochemically cross-reactive species present in brain (M_r= 205,000) is apparently due to the greater size of the PC12 cell tri- and tetraantennary complex oligosaccharides.     

Characterisation of a soluble trypsin fragment of GP130: a neuronal glycoprotein associated with the cytoskeleton

A neuronal glycoprotein (GP130) that is associated with the cytoskeleton [Ranscht et al: J Cell Biol 99:1803-1813, 1984] remains insoluble in 0.1 M NaOH, a property typical of integral membrane proteins. At present it is possible to solubilise and hence isolate GP130 only under denaturing conditions. However, a large fragment of apparent molecular weight 120K is released into solution by trypsin. The fragment corresponds to the extracellular region of the glycoprotein as shown by the fact that it is released from live cultures of chicken sympathetic neurons and by its retention of concanavalin A-binding activity. The soluble extracellular fragment has been purified using mild biochemical techniques, which are expected to retain its biological activity. Measurement of the sedimentation coefficient, Stokes radius, and frictional ratio in addition to metal shadowing of the fragment show that it has a molecular weight of about 120K and is asymmetric, probably rod-shaped with a long axis of more than 20 nm.     

The Dantu erythrocyte phenotype of the NE variety. I. Dodecylsulfate polyacrylamide gel electrophoretic studies

Red cell membranes from patient NE, Mr. Dantu and 16 additional Black individuals, positive for the low-frequency MNSs-system antigen Dantu, were studied by dodecylsulfate polyacrylamide gel electrophoretic techniques. The content of the major, blood group M- or N-active sialoglycoprotein (glycophorin A, GP A) was found to be decreased by about 57%. The blood group S- or s-active sialoglycoprotein (GP B) was decreased by about 51% in membranes from proven Dantu/U heterozygotes and not detectable in those from patient NE and other Dantu+U- individuals. Donor NE was shown to exhibit the genotype Dantu/u. Dantu-positive cells exhibit a proteinase-resistant GP B-GP A hybrid with an apparent molecular mass of 29 KDa whose intramembraneous and cytoplasmic domains were shown to be similar to those of GP A. The molar hybrid: GP A ratio in all cells was found to be about 2.4: 1, indicating that the NE variety of the Dantu phenotype is much more frequent than the Ph or MD types. The significance of an additional minor 'new' component (molecular mass 21 KDa) in Dantu+ membranes and the minor component J (molecular mass 22 KDa) occurring in normal and Dantu+U+ GP preparations, but not in those from Dantu+U- cells, has not been resolved. The apparent molecular mass of the anion channel protein (band 3) in all cells of the NE variety was shown to be decreased by about 3 KDa, due to a shortening of carbohydrate chains. This suggests that the hybrid, just like GP A, might form a complex with band 3.     

The regulatory subunit of neural cAMP-dependent protein kinase II represents a unique gene product

Although the major form of soluble cAMP-dependent protein kinase in bovine cerebral cortex can be classified as a type II kinase, the regulatory subunit (RII) can be distinguished from RII found in other tissues such as heart. Heart and brain RII were distinguished qualitatively by autophosphorylation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mobility of dephosphorylated heart RII shifted from an apparent Mr of 55,000 to 57,000 following autophosphorylation. In contrast, when RII purified from brain was autophosphorylated with [gamma-32P]ATP, two radiolabeled bands were visualized, a minor band (less than or equal to 20%) which migrated with an Mr of 57,000 similar to the heart protein and a band with Mr = 55,000 which did not shift its mobility in response to autophosphorylation. Brain RII was further distinguished from heart RII on the basis of cAMP binding. Millipore filtration and equilibrium dialysis indicated that 2 mol of cAMP bound/mol of RII in contrast to 4 mol/mol with heart RII. Immunological differences were also apparent. Radioimmunoassays using monoclonal antibodies to RII showed that the brain protein had less than 4% of the cross-reactivity of heart RII. Both immunoblotting and immunoprecipitation using monoclonal as well as serum antibodies established that the cross-reactivity in phosphorylated brain RII was associated exclusively with the 57,000 component that behaved like heart RII. The lack of cross-reactivity of neural RII with two different monoclonal antibodies targeted the hinge region of RII as an area where structural differences might be anticipated, and comparative sequence analysis of this region definitively established that the major form of RII in brain is a unique gene product from the RII expressed in heart.     

A comparison of the fibrinogen receptor distribution on adherent platelets using both soluble fibrinogen and fibrinogen immobilized on gold beads

The distribution of fibrinogen receptors was determined on the surface of adherent platelets using both direct labeling with the ligand fibrinogen which was immobilized on gold particles (Fg-Au) and indirect immunogold (Ig-Au) labeling of bound soluble fibrinogen identified with a rabbit polyclonal anti-fibrinogen antibody. Two distinctly different patterns of labeling were obtained and appeared to depend on whether solid phase fibrinogen (Fg-Au) or soluble phase released fibrinogen were bound to the membrane receptor. The membrane-bound Fg-Au reorganized in patterns that closely mimicked the organization of the underlying cytoskeleton. In approximately 18% of the adherent platelets, Fg-Au was seen in channels or vesicle-like structures lying deep to the platelet surface suggesting internalization into the open canalicular system and/or endocytosis. The labeling pattern obtained when identifying the location of membrane-bound soluble released fibrinogen by Ig-Au was diffuse and lacked the organizational patterns characteristic of Fg-Au. Unlike the Fg-Au probe, early dendritic platelets were heavily labeled by the soluble phase fibrinogen using the Ig-Au technique. Although the label covered the entire exposed platelet membrane in fully spread platelets, labeling over the peripheral web was more dense than that over the intermediate or granulomere zone. The diffuse organization and heavier peripheral distributional pattern of the glycoprotein IIb-IIIa (GP IIb-IIIa) receptor in fixed, adherent platelets, was also seen with the GP IIb-IIIa receptor-specific antibody AP-2. The binding of both the Fg-Au and Ig-Au were inhibited using the tetrapeptide Arg-Gly-Asp-Ser (RGDS) (93% and 98% inhibition, respectively), AP-2 (98% and 97%, respectively) and platelets from patients with Glanzmann's thrombasthenia (GT) (99% and 98%, respectively). The data presented provides the first report that receptor reorganization, following binding of fibrinogen, appears to be related to the state of the ligand. Substrate bound fibrinogen (i.e., Fg-Au or fibrinogen bound to another platelet) induces receptor translocation toward the platelet granulomere in a capping-like phenomenon. On the other hand, the binding of soluble released fibrinogen results in formation of microclusters and short linear arrays in a diffuse distribution but does not induce central movement of receptors. Furthermore, double labeling studies clarify that Fg-Au does not identify all available fibrinogen receptors as many are occupied by soluble released fibrinogen. The data presented provides an interesting new perspective on what constitutes an appropriate ligand-receptor stimulus sufficient to induce receptor reorganization.     

Isolation and Immunochemical Study of a Soluble Cerebellar Lectin Delineating Its Structure and Function

A procedure of sequential extractions of cerebellar tissue was set up, which allowed specific solubilization of endogenous lectins by mannose. Two cerebellar soluble lectins, CSL1 (Mr = 33,000) and CSL2 (Mr = 31,500), were isolated. They appeared to consist of structurally and immunologically related polypeptides chains. By immunoaffinity, another minor component (Mr = 45,000) was isolated. Immunological studies suggested that the minor component is the precursor of the two other, i.e., CSL1 and CSL2, subunits. CSL1 (mainly lysosomal) possesses an additional peptide compared with CSL2 (mainly cytoplasmic and extracellular), which seems to be implicated in the signal for secretion and release.     

Solubilization of the liver vasoactive intestinal peptide receptor. Hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein

Vasoactive intestinal peptide (VIP) receptors were solubilized using the nondenaturing detergent Triton X-100 after occupancy of rat liver membrane-bound receptors with 125I-VIP. Gel filtration and ultracentrifugation on sucrose density gradients revealed the existence in the soluble macromolecular fraction of two labeled components: a major (80%) heavy component and a minor (20%) light one. The two components exhibit the following hydrodynamic parameters: Stokes radius, 5.8 nm: s20,w, 5.98 s; Mr, 150,000; frictional ratio, 1.52 for the major; and Stokes radius, 3.0 nm: s20,w, 3.98 s; Mr = 52,000; frictional ratio, 1.12 for the minor component. The labeling of these components was specific in that it dramatically decreased when unlabeled VIP was added together with 125I-VIP. The pharmacological specificity was also assessed by using 10 nM histidylisoleucineamide (a VIP agonist). Many lines of evidence indicate that the light component (Mr = 52,000) is the VIP-receptor complex while the heavy component (Mr = 150,000) is a ternary complex consisting of VIP, the receptor, and a guanine nucleotide regulatory protein, probably Ns. GTP is required to dissociate 125I-VIP from the heavy component whereas it is ineffective on the light component. This effect is nucleotide specific. After cholera toxin-induced [32P]ADP ribosylation of liver membranes, a high peak of 32P radioactivity containing the alpha subunit (Mr = 42,000) of the Ns protein is coeluted with the heavy component on Sephacryl S-300. By mild urea (2 M) treatment, the heavy component is converted into the light without significant dissociation of 125I-VIP. When a Triton extract of membranes prelabeled with 125I-VIP is treated with dithiobis(succinimidyl propionate) subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a major band corresponding to Mr = 150,000. Alternatively, when prelabeled membranes are directly treated with the cross-linker, a major complex of Mr = 51,000 is observed. This may be related to different accessibility of the cross-linker to the site at which the receptor and the Ns protein interact in the two conditions. In conclusion, these data represent initial reports on the successful solubilization of functional VIP-receptor complexes and provide evidence for an interaction between liver VIP-receptor complexes and a GTP-binding protein.     

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. V. A small basic protein from culture supernatants is a potent T cell mitogen

Previous studies established that Mycoplasma arthritidis produces a soluble T cell mitogen (MAM), and that response of murine T cells to MAM is genetically restricted. MAM appeared predominantly in the supernatants of senescent cultures, but was not extracted in significant amounts from whole cells. A quantitative assay of MAM activity was devised. MAM formed noncovalent complexes with nucleic acids and uncharacterized high m.w. constituents of sera and of complex media. Partially purified MAM was adsorbed or denatured by glass and plastic surfaces. MAM was protease-labile, had pI greater than or equal to 9, and had Mr ca 15,000 according to gel filtration experiments. MAM was a very minor component of culture supernatant proteins, and even after 200- to estimated 5 X 10(4)-fold purification was not identified as a stainable or ultraviolet-absorbing entity in electrophoretigrams or chromatograms. It was estimated that MAM was half-optimally active at less than 1000th the half-optimal concentration of concanavalin A or phytohemagglutinin. Culture supernatants and highly purified MAM exhibited the same haplotype specificity (H-2k-dependent response) for stimulated proliferation of lymphocytes and for induction of interferon in vitro.     

Lipid and apolipoprotein distribution as a function of density in equine plasma lipoprotein

1. Equine lipoproteins were isolated from plasma by density gradient ultracentrifugation and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. VLDL and IDL were present at low concentration (0.2 mg/ml). Two apoB components of Mr corresponding to human apoB-100 and one apoB-48-like component were represented in VLDL fraction. 3. LDL-1 and LDL-2 subfractions have displayed an almost equal concentration (0.4 mg/ml). Two apoB-100-like components were the major apolipoproteins in each fraction. Small amounts of apoB-48-like component were detectable in LDL-1 and LDL-2. 4. HDL-2 represented a major class of equine lipoproteins (1.8 mg/ml). ApoA-1-like component was the dominant protein in HDL-1, HDL-2 and HDL-3. Dimeric apoA-II-like components were slightly represented in HDL subfractions. 5. HDL-3 displayed the same apolipoprotein pattern as HDL-1 and HDL-2, but two further minor proteins of Mr 20,000 and 14,000 were detected. 6. VHDL represented a minor class of lipoprotein (0.2 mg/ml). ApoA-I-like component was the major apolipoprotein of VHDL. Small amounts of apoA-IV-like, apoE-like, and Mr 55,000 protein were detectable. 7. ApoC-like of Mr lower than 10,000 was represented in all equine lipoprotein classes.     

Isolation and characterization of proteoglycans synthesized by cultured mesangial cells

Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells.     

Cholesterol accumulates in cell bodies,but is decreased in distal axons,of Niemann-Pick C1-deficient neurons

Niemann-Pick type-C (NPC) disease is characterized by a progressive loss of neurons and an accumulation of unesterified cholesterol within the endocytic pathway. Unlike other tissues, however, NPC1-deficient brains do not accumulate cholesterol but whether or not NPC1-deficient neurons accumulate cholesterol is not clear. Therefore, as most studies on cholesterol homeostasis in NPC1-deficient cells have been performed in fibroblasts we have investigated cholesterol homeostasis in cultured murine sympathetic neurons lacking functional NPC1. These neurons did not display obvious abnormalities in growth or morphology and appeared to respond normally to nerve growth factor. Filipin staining revealed numerous cholesterol-filled endosomes/lysosomes in NPC1-deficient neurons and the mass of cholesterol in cell bodies was greater than in wild-type neurons. Surprisingly, however, the cholesterol content of NPC1-deficient and wild-type neurons as a whole was the same. This apparent paradox was resolved when the cholesterol content of NPC1-deficient distal axons was found to be less than of wild-type axons. Cholesterol sequestration in cell bodies did not depend on exogenously supplied cholesterol since the cholesterol accumulated before birth and did not disperse when neurons were cultured without exogenous cholesterol. The altered cholesterol distribution between cell bodies and axons suggests that transport of cholesterol, particularly that synthesized endogenously, from cell bodies to distal axons is impaired in NPC1-deficient neurons.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)