Malka G. Scher

Two populations of β-spectrin in rat skeletal muscle

We use immunoblotting, immunoprecipitation, and centrifugation in sucrose density gradients to show that the product of the erythrocyte β-spectrin gene in rat skeletal muscle (muscle β-spectrin) is present in two states, one associated with fodrin, and another that is not associated with any identifiable spectrin or fodrin subunit. Immunofluorescence studies indicate that a significant amount of β-spectrin without α-fodrin is present in the myoplasm of some muscle fibers, and, more strikingly, at distinct regions of the sarcolemma. These results suggest that α-fodrin and muscle β-spectrin associate in muscle in situ, but that some muscle β-spectrin without a paired α-subunit forms distinct domains at the sarcolemma. Cell Motil. Cytoskeleton 37:7–19, 1997.

Subcellular sites of enzymes catalyzing the phosphorylation-dephosphorylation of dolichol in the central nervous system☆

The subcellular locations of several enzymes involved in dolichyl monophosphate (Dol-P) metabolism in brain have been investigated. Dolichol kinase is highly enriched in a heavy microsomal fraction from calf brain, while 71% of the Dol-P phosphatase activity was recovered with the light microsomes. Lower amounts of the phosphatase activity were also found in the heavy microsomal, mitochondrial-lysosomal, and synaptic plasma membrane fractions. Since the light microsomal fraction also contained substantial acetylcholinesterase activity, an axon plasma membrane marker, an axolemma-enriched fraction, was prepared from rat brain by a second procedure. A comparison with microsomal and mitochondrial-lysosomal fractions revealed that the axolemma-enriched fraction contained the highest specific activity of Dol-P phosphatase, indicating that the enzyme was present in the axon plasma membrane. The tunicamycin-sensitive UDP-N-acetylglucosamine:Dol-P N- acetylglucosaminylphosphotransferase , glucosyl- phosphoryldolichol (Glc-P-Dol) synthase, Glc-P-Dol:oligosaccharide glucosyltransferase, and the oligosaccharyltransferase were all found predominantly in the heavy microsomes. These results indicate that the enzymes responsible for the initiation and termination of biosynthesis, as well as the transfer of dolichol-linked oligosaccharides, reside in the rough endoplasmic reticulum (ER) of central nervous tissue. Evidence that at least some Dol-P molecules formed by dolichol kinase are accessible to multiple glycosyltransferases in the rough ER of brain is also presented.

Enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate by calf brain membranes

Abstract Calf brain membranes have been shown to enzymatically dephosphorylate endogenous and partially purified, exogenous dolichyl [ 32 P]monophosphate. The properties and specificity of the dolichyl monophosphatase activity have been studied by following the release of [ 32 P]phosphate from exogenous dolichyl [ 32 P]monophosphate added in a dispersion with Triton X-100. The calf brain phosphatase (1) is inhibited by Mn 2+ , Mg 2+ , Ca 2+ , fluoride, and phosphate; (2) exhibits a neutral pH optimum; and (3) has an apparent K m of 200 μ m for dolichyl monophosphate. Dolichyl monophosphatase activity can be distinguished from phosphatidate phosphatase on the basis of their responses to fluoride and phosphate. Based on differential thermolability and the effects of divalent cations and EDTA, the calf brain dolichyl monophosphatase can also be discriminated from the general phosphatase activity assayed with p -nitrophenyl phosphate. Dolichyl monophosphatase activity can be solubilized by treating microsomes with Triton X-100. The enzymatic dephosphorylation of exogenous dolichyl [ 32 P]monophosphate catalyzed by particulate and detergent-solubilized preparations is negligibly affected by equimolar concentrations of ATP and an assortment of phosphomonoesters, including phosphatidic acid and hexadecyl phosphate. A reduction of approximately 40% in dolichyl monophosphatase activity is observed in the presence of equimolar amounts of retinyl monophosphate. Overall, these results represent good evidence for the presence of a neutral polyisoprenyl monophosphatase in central nervous tissue.

Glucosylphosphoryldolichol: Role as a glucosyl donor in the biosynthesis of an oligosaccharide lipid intermediate by calf brain membranes

When membrane preparations from calf brain are incubated with exogenous [ 14 C]-glucosylphosphoryldolichol, [ 14 C]glucose is enzymatically transferred into an endogenous oligosaccharide lipid. Efficient transfer was achieved by dispersing the lipophilic glucosyl donor in Triton X-100. The transfer of [ 14 C]glucose from exogenous [ 14 C]glucosylphosphoryldolichol into [Glc-[ 14 C]oligosaccharide lipid did not require the addition of a divalent cation, and occurred in the presence of EDTA. The inclusion of Ca 2+ in the reaction mixture resulted in a lower level of [Glc- 14 C]oligosaccharide lipid recovery and the incorporation of label into glycoprotein. If Ca 2+ is added following a 10-min preincubation of membranes with exogenous [ 14 C]glucosylphosphoryldolichol, label is lost from the oligosaccharide lipid fraction and there is a corresponding appearance of label in glycoprotein. The [Glc- 14 C]-oligosaccharide lipid glycosylated via [ 14 C]glucosylphosphoryldolichol in brain membranes is recovered by extraction with CHCl 3 -CH 3 OH-H 2 O (10:10:3). The chromatographic behavior of the labeled oligosaccharide lipid on DEAE-cellulose is similar to that of the oligosacharide lipids in which the oligosaccharide units are bound to dolichol by a pyrophosphate linkage. Consistent with this proposed structure, the [Glc- 14 C]oligosaccharide is released from the carrier lipid by treatment with 0.1 n HCl in 80% tetrahydrofuran at 50°C for 20 min. The free [Glc- 14 C]oligosaccharide unit contains approximately 10 glycose residues based on gel filtration analysis. The presence of two N -acetylated sugars is indicated by the formation of two positively charged products by treatment with strong base and the subsequent electrophoretic neutralization under conditions used for N -acetylation. The labeled product recovered in the delipidated membrane residue following incubation of calf brain membranes with exogenous [ 14 C]glucosylphosphoryldolichol in the presence of Ca 2+ can be solubilized with 1% sodium dodecyl sulfate-0.1% mercaptoethanol (100°C, 2 min). The solubilized product is resolved into two large molecular weight fractions by chromatography on a Bio-Gel A-1.5m column eluted with a buffer containing 0.2% sodium dodecyl sulfate-0.1% mercaptoethanol. Both high molecular weight fractions are converted to low molecular weight products by digestion with Pronase or trypsin. The labeled glycopeptide fraction produced by Pronase digestion is eluted as a single peak on Bio-Gel P-6 and appears to be slightly larger than the glucosylated oligosaccharide released from the carrier lipid by mild acid treatment. Strong acid hydrolysis (3 n HCl, 100°C, 6 h) of the [ 14 C]-glycopeptides released a single radioactive product that cochromatographed with authentic glucose. The electrophoretically neutral [Glc- 14 ]glycopeptide fractions by treatment with mild alkali, indicating that the [Glc- 14 C]oligosaccharide units are probably attached to the polypeptide chains by an N -glycosidic linkage. These results show that in calf brain membranes at least one glucose residue in a glucosylated oligosaccharide lipid is acquired via glucosylphosphoryldolichol, and strongly suggest that in the presence of Ca 2+ the glucosylated oligosaccharide unit can be enzymatically transferred from its carrier lipid into glycoprotein

Lipolytic cleavage of dolichyl oleate catalyzed by calf brain membranes

Abstract Calf brain membranes catalyze the lipolytic cleavage of dolichyl [ 14 C]oleate added as an aqueous dispersion in Triton X-100. The enzymatic release of [ 14 C]oleate from the dolichyl ester is not affected by divalent cations or EDTA, but the lipase activity is inhibited by iodoacetamide and pHMB. The amount of [ 14 C]oleate released is dependent on the time of incubation, the amount of membrane protein added and the concentration of the radiolabeled lipid substrate. Dolichyl ester hydrolase activity exhibits a pH optimum of 7.5, distinguishing this lipase activity from cholesteryl ester hydrolase (5.0–5.5) and triolein hydrolase (5.0) activity associated with the same membrane preparations. The enzymatic hydrolysis of dolichyl [ 14 C]oleate is also partially inhibited by oleate and free dolichol, possibly by end-product inhibition.

Stabilization of immobilized lectin columns by crosslinking with glutaraldehyde

Procedures to purify membrane proteins usually require the use of detergents and often include affinity chromatography on lectin columns. Some detergents, especially denaturing detergents such as sodium dodecyl sulfate (SDS), can interfere with affinity chromatography by inactivating the bound lectin or by eluting it from the column together with the material of interest. We have developed a procedure that stabilizes lectin-column matrices by crosslinking with glutaraldehyde. This procedure does not impair the binding capacity of the immobilized lectin. It permits subsequent elution by SDS of bound glycoproteins without coelution of lectin subunits.

Enzymatic Glucosylation of Dolichyl Monophosphate Formed Via Cytidine Triphosphate in Calf Brain Membranes

When calf brain membrane preparations containing endogenous dolichyl [32P]monophosphate (Dol‐32P), prelabeled enzymatically by [γ‐32P]‐CTP, are incubated with unlabeled UDP‐glucose, the formation of a mild acid‐labile [32P]phosphoglucolipid is observed. The biosynthesis of the [32P]phosphoglucolipid is dependent on the concentration of UDP‐glucose added, and no [32P]phosphoglycolipid appeared when UDP‐glucose was replaced by ADP‐glucose, UDP‐xylose, UDP‐galactose, UDP‐mannose, or UDP‐glucuronic acid. The 32P‐labeled product formed by the UDP‐glucose‐dependent reaction is chemically and chromatographically identical to glucosylphosphoryldolichol. Several enzymatic parameters of the glucosylation of the specific pool of Dol‐P, synthesized by the CTP‐mediated kinase, and the total available pool of Dol‐P have been compared by a double‐label assay utilizing endogenous, prelabeled Dol‐32P and UDP‐[3H]glucose as substrates.