Purification of a sarcoplasmic reticulum protein that binds Ca2+ and plasma lipoproteins

A protein in the sarcoplasmic reticulum of rabbit skeletal and cardiac muscle was identified because of its ability to bind 125I-labeled low density lipoprotein (LDL) with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, referred to as the 165-kDa protein, is restricted to striated muscle. It was not detected in 14 other tissues, including several that contain smooth muscle, but it appears in rat L6 myoblasts when they differentiate into myocytes. Immunofluorescence and immunoelectron microscopic studies revealed that the protein is present throughout the sarcoplasmic reticulum and the terminal cisternae. It binds 45Ca2+ on nitrocellulose blots and stains metachromatically with Stains-all, a cationic dye that stains Ca2+-binding proteins. It does not appear to be a glycoprotein, and it appears slightly larger than the 160-kDa glycoprotein previously described in sarcoplasmic reticulum. The 165-kDa protein binds LDL, beta-migrating very low density lipoprotein, and a cholesterol-induced high density lipoprotein particle that contains apoprotein E as its sole apoprotein with much higher affinity than it binds high density lipoprotein. The protein is stable to boiling and to treatment with sodium dodecyl sulfate, but it becomes sensitive to these treatments when its cystine residues are reduced and alkylated. The protein was purified 1300-fold to apparent homogeneity from rabbit skeletal muscle membranes. It differs from the cell surface LDL receptor in that 1) its apparent molecular weight is not changed by reduction and alkylation; 2) it is present in Watanabe-heritable hyperlipidemic rabbits, which lack functional LDL receptors; 3) binding of lipoproteins is not inhibited by EDTA; and 4) it is located within the lumen of the sarcoplasmic reticulum where it has no access to plasma lipoproteins. It is unlikely that this protein ever binds lipoproteins in vivo; however, its lipoprotein binding activity has facilitated its purification to homogeneity and suggests that this protein has unusual structural features. The role of the 165-kDa protein in Ca2+ homeostasis in the sarcoplasmic reticulum, if any, remains to be determined.     

Structural and enzymatic studies of the T4 DNA replication system. I. Physical characterization of the polymerase accessory protein complex

In this study, we have investigated the structural and physical properties of the bacteriophage T4 DNA polymerase accessory proteins. We find that T4 gene 44 and 62 proteins associate to form a tight, highly homogeneous complex, containing four gene 44 protein subunits and one gene 62 protein subunit. The molecular mass of the complex is 163,700 daltons. Sedimentation results suggest that the complex is quite asymmetric, with a prolate ellipsoid axial ratio of about 5:1. This protein complex is known to carry a DNA-dependent ATPase activity; we show by photoaffinity labeling that the ATP-binding sites reside in the gene 44 protein subunits of the complex. Equilibrium sedimentation and chemical cross-linking studies indicate that the T4 gene 45 protein self-associates to form a trimer in solution. This trimer species also appears to be quite asymmetric, showing an axial ratio for a prolate ellipsoid of about 6:1, assuming normal hydration.     

O-linked glycosylation of rat renal gamma-glutamyltranspeptidase adjacent to its membrane anchor domain

Large domains rich in serine and threonine, that are likely to exhibit clusters of O-linked oligosaccharides, have been reported adjacent to the anchor of several cell surface proteins. No such domain is evident in the primary sequence of rat renal gamma-glutamyltranspeptidase. However, papain treatment of the amphipathic enzyme (Triton-purified gamma-glutamyltranspeptidase, T gamma GT), pretreated with galactose oxidase and NaB3H4 (Frielle, T., and Curthoys, N. P. (1983) Biochemistry 22, 5709-5714), yields the hydrophilic enzyme (papain-treated Triton-purified gamma-glutamyltranspeptidase, PT gamma GT) and a labeled peptide which contains both the amino-terminal membrane anchor and the sequence Pro27-Thr28-Thr29-Ser30. Since [3H]galactose was identified in this peptide, the presence of O-linked oligosaccharides was investigated. Carbohydrate analysis is consistent with the presence of two simple O-linked oligosaccharides on T gamma GT and one on PT gamma GT. Lectin blot analysis of T gamma GT and PT gamma GT was carried out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The small subunits of both T gamma GT and PT gamma GT and the large amphipathic subunit of T gamma GT all react with the peanut agglutinin lectin, but the large subunit of PT gamma GT exhibits no such reactivity. The reactivity with PNA is consistent with the presence of one oligosaccharide with the structure galactose beta 1-3N-acetylgalactosamine alpha 1-Ser/Thr attached to each subunit of T gamma GT. The papain-sensitivity of the oligosaccharide from the larger subunit is consistent with O-glycosylation at the Thr28-Thr29-Ser30 sequence. The results of lectin blot analysis with wheat germ agglutinin imply that the content of N-linked oligosaccharides is unaffected by papain treatment of the transpeptidase. These data represent the first direct evidence for O-glycosylation of a microvillar hydrolase at a site immediately adjacent to the membrane anchor and indicates that even small clusters of Thr and Ser can be O-glycosylated. Isolated O-linked oligosaccharides may have functional significance since single Ser and Thr residues are consistently found near the membrane anchor of many cell surface proteins.     

Construction of the endoplasmic reticulum

To study the construction of the ER, we used the microtubule-disrupting drug nocodazole to induce the complete breakdown of ER structure in living cells followed by recovery in drug-free medium, which regenerates the ER network within 15 min. Using the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide to visualize the ER, we have directly observed the network construction process in living cells. In these experiments, the ER network was constructed through an iterative process of extension, branching, and intersection of new ER tubules driven by the ER motility previously described as tubule branching. We have tested the cytoskeletal requirements of this process. We find that newly formed ER tubules are aligned with single microtubules but not actin fibers or vimentin intermediate filaments. Microtubule polymerization preceded the extension of ER tubules and, in experiments with a variety of different drugs, appeared to be a necessary condition for the ER network formation. Furthermore, perturbations of the pattern of microtubule polymerization with microtubule-specific drugs caused exactly correlated perturbations of the pattern of ER construction. Induction of abnormally short, nonintersecting microtubules with 20 microM taxol prevented the ER network formation; ER tubules only extended along the few microtubules contacting the aggregated ER membranes. This requirement for a continuous network of intersecting microtubules indicates that ER network formation takes place through the branching and movement of ER membranes along microtubules. Cytochalasin B had no apparent effect on the construction of the ER network during recovery, despite apparently complete disruption of actin fibers as stained by phalloidin. Blockage of protein synthesis and disorganization of intermediate filaments with cycloheximide pretreatment also failed to perturb ER construction.     

Kinetics of insulin internalization and processing in adipocytes: effects of insulin concentration

We have studied the effect of insulin concentration on the kinetics of insulin internalization and efflux in isolated rat adipocytes. To determine internalization rates adipocytes were incubated with 125I-insulin at 37 degrees C; and at frequent, early time points surface-bound and intracellular insulin were quantitated. Surface-bound and intracellular insulin were discriminated by the sensitivity of the former to rapid dissociation by a pH 3.0 buffer at 4 degrees C. From this data the endocytotic (internalization) rate constant (ke) was calculated for six insulin concentrations ranging from 0.3 to 100 ng/ml. Ke was found to decrease in an insulin concentration-dependent manner (P less than .001). Thus, values for ke were 0.121 +/- 0.006 min-1 versus 0.074 +/- 0.011 min-1 at 0.3 ng/ml and 100 ng/ml, respectively. The decrease in ke did not parallel insulin concentration-dependent changes in insulin receptor affinity indicating it was not the result of an inability of low affinity receptors to be internalized. The kinetics of insulin efflux were determined by loading various concentrations of 125I-insulin into the adipocyte interior, washing away surface-bound and extracellular insulin, and then monitoring the subsequent efflux of pre-loaded insulin into medium that contained the same concentration of insulin used in the loading step. The overall rate of efflux was independent of insulin concentration. In summary, these results show that at high insulin concentrations the efficiency of insulin internalization is impaired. In contrast, the rate of insulin efflux is unaffected.     

Serotonin uptake and configurational change of bovine pulmonary artery smooth muscle cells in culture

Although it is well known that endothelial cells transport serotonin (5-HT) from extracellular to intracellular locations, it has been generally assumed that smooth muscle cells do not accumulate 5-HT but, rather, respond to 5-HT through a receptor activity unrelated to uptake of this amine or via stimulation of endothelial-derived relaxing factor. In the present study smooth muscle cells (PASMC), isolated and cultured from bovine pulmonary artery, were evaluated for 5-HT uptake under a variety of conditions. 5-HT uptake was linear up to 15 min and the rate was seven- to eightfold higher than that by bovine pulmonary artery endothelial cells. There was intracellular metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA). The uptake was inhibited by exposure to 4 degrees C, absence of Na+ from the medium, and agents such as imipramine, verapamil, ketanserin, and methiothepin. Like that of endothelial cells, 5-HT uptake by PASMC was stimulated by exposure of cells to anoxia for 24 hr. Unlike endothelial cells that showed no morphological changes, PASMC at early passage showed dendritic formation after 30-60 min exposure to 5-HT at a concentration as low as 10(-8) M. Although this configurational change in response to 5-HT was lost with passage of cells, transport of 5-HT by these cells was retained. The configurational change was blocked by agents that inhibited 5-HT uptake, such as imipramine, verapamil, ketanserin, and methiothepin; it was unaffected by inhibitors of protein kinase C, phospholipase C, and calmodulin or absence of Ca2+ from the medium. We conclude that PASMC, as well as endothelial cells, accumulate 5-HT; there appears to be a close relationship between 5-HT uptake and configurational change of early passaged PASMC in culture. The factor(s) required for the configurational change are absent in endothelial cells and lost during passage of PASMC.     

An improved method for preparation, stabilization, and storage of house fly (Diptera: Muscidae) microsomes

An improved method for preparation and storage of insect microsomes from house fly, Musca domestica L., abdomens was developed. Microsomes were prepared in phosphate buffer fortified with glycerol, dithiothreitol, ethylenediaminetetraacetic acid, phenylmethylsulfonyl fluoride, and 1-phenyl-2-thiourea. No cytochrome P-420 was observed when abdomens were isolated by our method. No measurable loss of cytochrome P-450, cytochrome b5 or NADPH-cytochrome c (P-450) reductase levels, or methoxyresorufin O-demethylation, 7-ethoxycoumarin O-deethylation, or aryl hydrocarbon hydroxylation activities occurred when a diluted suspension (protein concentration of 2 mg/ml) of microsomes was stored at -80 degrees C for at least 2 mo.     

Elemental composition and food intake of Phialidium hydromedusae in the laboratory

Elemental composition and feeding rate of hydromedusae Phialidium sp. on copepods were studied in the laboratory. Regression equations for both mature and immature medusae allowed the estimation of their dry weight (DW), total C and N content as a function of their diameter. The mean C content as percentage of the DW varied from 13.13% ( ) for the immature to 19.38% (5.68) for the mature individuals. The mean N content is 4.03% (2.49) of DW of immatures and 5.85% (2.70) of the matures. Ingestion rate of Phialidium sp. fed on copepods (200–500 μm) increased with prey density but reached a maximum at high prey concentrations. A maximum ingestion rate of 8.55 (1.6) copepods · medusa ⁻¹ · h⁻¹ was reached for prey concentrations of > 140 copepods · 1 ⁻¹ for both immature and mature medusae. Maximum daily consumption of prey weight varied from 1.41 to 978% C body weight for mature medusae and from 2.90 to 975% for the immature individuals.     

Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. II. Suppression of disease and in vitro immune responses is mediated by antigen-specific CD8+ T lymphocytes

We have previously demonstrated that the oral administration of guinea pig myelin basic protein (MBP) protects Lewis rats against the induction of experimental autoimmune encephalomyelitis (EAE) when subsequently immunized with guinea pig MBP in CFA. In addition, animals made orally tolerant to MBP also have diminished proliferative and antibody responses to MBP, but not to other Ag. Nonetheless, the mechanism of oral tolerance to MBP in the EAE model remains undefined. In the present study, we report that T cells isolated from the spleen and mesenteric lymph nodes of MBP orally tolerized animals can adoptively transfer protection against EAE. Furthermore, these T cells are of the CD8+ subclass. In addition, CD8+ T cells from MBP orally tolerized animals also suppress in vitro proliferative responses and antibody responses to MBP in an Ag-specific fashion. These results demonstrate that active cellular mechanisms are initiated after oral administration of an autoantigen that can down-regulate an experimental autoimmune disease and provide the basis for the isolation and characterization of the cells mediating both in vivo and in vitro suppression.     

Superinduction of the murine B cell Fc epsilon RII by T helper cell clones. Role of IL-4

Th cell clones are known to induce an IL-4 dependent polyclonal IgE synthesis. Because IL-4 can induce the expression of the low affinity FcR for IgE (Fc epsilon RII) the ability of Th cell clones to induce Fc epsilon RII on purified splenic B cells was analyzed. It was found that a TH2 clone could cause a 50- to 100-fold superinduction of Fc epsilon RII after 2 days in culture; after 3 days, the Fc epsilon RII levels had almost returned to base line. The superinduction was inhibited by an anti-IL-4 antibody, 11B11, indicating its dependence on IL-4. A TH1 clone could cause a modest (four fold) induction of Fc epsilon RII, and this induction was not influenced by 11B11. A similar Fc epsilon RII induction was seen when using the supernatant from activated TH1 cells. The component(s) causing this relatively low level Fc epsilon RII induction is not known; a variety of known lymphokines were tested, and only IL-4 demonstrated any capacity for Fc epsilon RII induction on LPS-activated B cells. Addition of rIL-4 at concentrations of 400 U/ml or greater to the TH1 culture was sufficient to cause a Fc epsilon RII superinduction similar to that seen with the TH2 clone, while 40 U/ml was not. In order to determine a potential role for the Fc epsilon RII or its soluble fragment on the IgE synthesis mediated by TH2, a monoclonal anti-Fc epsilon RII, B3B4, was added to the culture. The addition of B3B4 did not have an influence on IgE levels in this system.