Butyrophilin,an apical plasma membrane-associated glycoprotein characteristic of lactating mammary glands of diverse species

Lipid globule membranes were isolated from human and bovine milk and from the milk of sheep, goat, pig, rat and guinea pig, and their polypeptide compositions were analyzed. The major polypeptides with molecular weights similar to that of bovine butyrophilin were separated by gel electrophoresis, isolated and characterized with respect to isoelectric point, molecular weight, immunological cross-reactivity and peptide composition after proteolytic cleavage. We show that in all species examined these proteins are similar to bovine butyrophilin in (i) their relative insolubility in buffers of low and high ionic strength and in non-denaturing detergents, (ii) the occurrence of several isoelectric variants, and (iii) patterns of peptides obtained by protease digestion. It is concluded that closely related proteins are major constituents of the cytoplasmic coat structures associated with milk lipid globule membranes of many species, and we propose the name butyrophilins for this group of proteins. Bovine and human butyrophilins are glycosylated with relatively large amounts of glucosamine, mannose, glucose and galactose but little fucose, sialic acids or galactosamine. Most if not all of the sugar residues are associated with an acetone-soluble peptide fragment of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 12 000–16 000 focusing at about pH 4.0. We suggest that this fragment contains a membrane-spanning peptide sequence and is involved in the attachment of the cytoplasmic coat to the membrane of the milk lipid globule.     

Mechanism of phosphorylation in the lumen of the Golgi apparatus. Translocation of adenosine 5'-triphosphate into Golgi vesicles from rat liver and mammary gland

The occurrence of phosphorylated secretory proteins such as caseins and vitellogenin and the recent characterization of phosphorylated proteoglycans, in the xylose and protein core, has raised the question of where in the cell and how this phosphorylation occurs. Previous studies have described a casein kinase activity in the lumen of the Golgi apparatus and this organelle as the site of xylose addition to the protein core of proteoglycans. We now report the translocation in vitro of ATP into the lumen of rat liver and mammary gland Golgi vesicles which are sealed and have the same membrane topographical orientation as in vivo. The entire ATP molecule was translocated into the lumen of the Golgi vesicles; this was established by using ATP radiolabeled with tritium in the adenine and gamma-32P. Translocation was temperature dependent and saturable, with an apparent Km of 0.9 microM and Vmax of 58 pmol/mg protein/min. Preliminary evidence suggests that translocation of ATP into the vesicles' lumen is coupled to exit of AMP from the lumen. Following translocation of ATP into the lumen of the vesicles, proteins were phosphorylated.     

Golgi apparatus isolation and use in cell-free systems

Summary Recent advances in understanding the molecular mechanisms of membrane traffic to and through the Golgi apparatus have been predicated in large measure on the use of permeabilized animal cells, and on completely cell-free systems. These systems have included those addressing inter-Golgi apparatus membrane traffic, endoplasmic reticulum to Golgi apparatus traffic, and endocytotic events. Development of cell-free systems depends on the use of isolated fractions. Specificity is often achieved by using a compartment-specific assay so that the fractions employed can be very crude. More recently cell-free systems also have evolved which employ highly purified and well-characterized cell fractions. The latter may be utilized in the absence of a compartment-specific assay but may require employment of compartment-specific assays for validation. Central to development of cell-free systems for membrane analysis has been the availability of isolated Golgi apparatus, first from plants and later from animal tissues and cells. A major advantage of cell-free systems is that they are most clearly amenable to the investigation of molecular mechanisms of membrane trafficking.Dedicated to Hilton H. Mollenhauer on the occasion of his retirement     

Direct resolution of enantiomers of basic imidazol-2-yl-substituted alcohols by gas chromatography on chirasil-val

The direct resolution of enantiomers of a series of imidazol-2-yl-substituted alcohols has been achieved by gas chromatography on a well-deactivated fused-silica capillary column, coated with L -Chirasil-Val as the chiral stationary phase. The separation of these basic compounds is accomplished without exaggerated peak tailing. Compared to simpler alcohols the resolution factors (α) observed are extraordinarily large. While the imidazolyl substituent may contribute to the mechanism of the chiral discrimination, the crucial interaction is assumed to be through the hydroxy group, based on the observation that the resolution factors for the corresponding O-acetyl derivatives are markedly reduced. © 1993 Wiley-Liss, Inc.     

Extracellular polysaccharide of Nostoc commune (Cyanobacteria) inhibits fusion of membrane vesicles during desiccation

Cells of the cyanobacterium Nostoc commune secrete a complex, high molecular weight, extracellular polysaccharide (EPS) which accumulates to more than 60% of the dry weight of colonies. The EPS was purified from the clonal isolate N. commune DRH1. The midpoint of the membrane phase transition (T_m) of desiccated cells of N. commune CHEN was low (T_{m dry} = 8 °C) and was comparable to the T_m of rehydrated cells((T_m)_H20 = 6 °C). The EPS was not responsible for the depression of T_m. However, the EPS, at low concentrations, inhibited specifically the fusion of phosphatidylcholine membrane vesicles when they were dried in vitro at0% relative humidity (−400 MPa). Low concentrations of a trehalose:sucrose mixture, in a molar ratio which corresponded with that present in cells in vivo, together with small amounts of the EPS, were efficient in preventing leakage of carboxyfloroscein (CF) from membrane vesicles. Freeze-fracture electron microscopy resolved complex changes in the structure of the EPS and the outer membrane in response to rehydration of desiccated cells. The capacity of the EPS to prevent membrane fusion, the maintenance of a low T_{m dry} in desiccated cells, and the changes in rheological properties of the EPS in response to water availability, constitute what are likely important mechanisms for desiccation tolerance in this cyanobacterium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ganglioside biosynthesis. Characterization of CMP-N-acetylneuraminic acid : lactosylceramide sialyltransferase in Golgi apparatus from rat liver.

An enzyme that transfers sialic acid from GMP-sialic acid to lactosylceramide was concentrated 40-50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents as dispersing agents. Of the numerous detergents tested, the combination Tween 80-Triton CF-54 (1 : 2, w/w) was the most effective in stimulating the reaction. Two apparent pH optima, at 6.35 and 5.5, were observed. The enzyme showed no requirement for a divalent cation. The Km values calculated for CMP-N-acetylneuraminic acid and lactosylceramide were 2.7 - 10(-3) and 1.3 - 10(-4) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane. The newly synthesized GM3, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.     

Neuronal repair and avoidance behavior in the flatworm,Notoplana acticola

In Notoplana avoidance behavior is lost after bisection of the brain or removal of one of its lobes. Behavioral recovery usually occurs within 3–10 days. Recovery of individuals may be gradual or abrupt. Grouped data shows gradual linear repair of turning behavior. Most animals with all connectives between the two lobes of the brain severed recovered preoperative responses, while those with one lobe of the brain removed averaged about 60% of the preoperative level of response. Some individuals in both groups recovered completely. Histological evidence of neuronal repair was found in all animals. Where the lobes of the brain were separated, connectives between them appeared to re-form. In worms with one lobe of the brain removed, the nerves disconnected by the excision joined the remaining lobe. Action potentials are conducted across repaired tissue in both split-brain and half-brain worms in both seawater and Mg²⁺-rich solutions. CNS repair appears to involve functional synaptic contacts. Notoplana does not replace ganglionic tissue but does compensate adequately for CNS damage.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca/Mg)-ATPase, and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Dolichyl phosphate phosphatase from Tetrahymena pyriformis.

A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound.