scholarly journals Further characterization of the acid-soluble phosphoprotein (SDS/PAGE apparent molecular mass of 22 kDa) in rat fat-cells by peptide sequencing and immuno-analysis: effects of insulin and isoprenaline

1995 ◽  
Vol 306 (1) ◽  
pp. 135-139 ◽  
Author(s):  
T A Diggle ◽  
G B Bloomberg ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Molecular Mass ◽  
Peptide Sequencing ◽  
Apparent Molecular Mass ◽  
Epididymal Adipose Tissue ◽  
Fat Cells ◽  
Western Blots ◽  
Antibody Preparation ◽  
Brown Adipose ◽  
Sds Page

1. Earlier studies have shown that exposure of fat-cells to insulin results in the rapid increased phosphorylation of an acid-soluble protein which migrates as a doublet on SDS/PAGE with an apparent molecular mass of close to 22 kDa; agents such as isoprenaline, which increase cell concentrations of cyclic AMP, also increase phosphorylation, but to a lesser extent [Belsham, Brownsey, Hughes and Denton (1980) Diabetologia 18, 307-312; Diggle and Denton (1992) Biochem. J. 282, 729-736]. 2. The protein has been purified from rat epididymal adipose tissue, and the sequences of six tryptic peptides were determined. All six peptides are present in the deduced sequence of a protein of similar properties, designated PHAS-I by Hu, Pang, Kong, Velleca and Lawrence [(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3730-3734]. Hence the proteins are the same or extremely similar. 3. A rabbit anti-peptide antibody has been raised against one of the peptides (AGGDESQFEMD). The antibody was found to be highly specific for the phosphorylated and non-phosphorylated forms of the acid-soluble 22 kDa protein in Western blots and by immunoprecipitation. Studies with the antibody preparation have shown that both phosphorylated and non-phosphorylated forms of the protein appear to be exclusively located in the cytoplasm, and that exposure of cells to isoprenaline causes increased phosphorylation of the same acid-soluble 22 kDa protein as does insulin treatment. 4. Western blots carried out with the antibody preparation indicate that the protein is also present in other insulin-sensitive tissues, including liver, skeletal muscle, heart and brown adipose tissue. The protein was also detected in lung and spleen, but not brain and kidney. It is concluded that the protein may play an important role in some of the actions of insulin.

scholarly journals Anti-Obesity Effect of Galacto-Oligosaccharides in Obese Rats

2021 ◽  
Author(s):  
Shang Kong ◽  
Xingjun Huang ◽  
Hua Cao ◽  
Yan Bai ◽  
Qishi Che ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Fat Body ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Protective Role ◽  
Lipoprotein Cholesterol ◽  
Fat Cells ◽  
Expression Levels ◽  
Brown Adipose ◽  
Obese Rats

Abstract Background: Galacto-oligosaccharides (GOS) is a commonly used as a prebiotic with a variety of metabolic benefits. Whether GOS plays a protective role in obesity is still unknown. Here we demonstrated that GOS possesses an anti-obesity activity by promoting adipose tissue browning and thermogenesis. Results: Our results showed that GOS effectively slow weight gain of diet-induced obese (DIO) rats without affecting energy intake. GOS significantly suppressed the hypertrophy and hyperplasia of white adipose tissue (WAT), as well as markedly lessened the ratio of fat pad to fat body. Consistently, GOS significantly improved serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels, which indicated an appropriate weight loss activity of GOS. Interestingly, GOS also significantly increased the expression levels of browning proteins (UCP1, PPARγ, PGC1α and PRMD16) both in the WAT and brown adipose tissue (BAT). We further found that GOS markedly increased the expression levels of LXRα, PPARα, LDLR and CYP7A1 proteins in the liver of obese rats. Conclusions: Taken together, we concluded that GOS inhibits obesity by accelerating the browning of white fat cells and the thermogenesis of brown fat cells, moreover GOS improves host lipid homeostasis by promoting cholesterol catabolism.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals An integral glycoprotein associated with the membrane attachment sites of actin microfilaments.

1985 ◽  
Vol 101 (3) ◽  
pp. 785-801 ◽  
Author(s):  
A A Rogalski ◽  
S J Singer
Keyword(s):  
Smooth Muscle ◽  
Chick Embryo ◽  
Molecular Mass ◽  
Apparent Molecular Mass ◽  
Reduced Form ◽  
Actin Microfilaments ◽  
Attachment Sites ◽  
Sds Page ◽  
Membrane Attachment ◽  
Embryo Fibroblasts

An integral membrane protein associated with sites of microfilament-membrane attachment has been identified by a newly developed IgG1 monoclonal antibody. This antibody, MAb 30B6, was derived from hybridoma fusion experiments using intact mitotic cells of chick embryo fibroblasts as the immunization vehicle as well as the screening probe for cell surface antigens. In immunofluorescent experiments with fixed cells, MAb 30B6 surface labeling is uniquely correlated with microfilament distributions in the cleavage furrow region of dividing chick embryo fibroblasts and cardiac myocytes in culture. The MAb 30B6 antigen in addition is associated with microfilament-membrane attachment sites in interphase fibroblasts at the dorsal surface, the adhesion plaque region at the ventral surface, and at junction-like regions of cell-cell contact. It is also found co-localized with the membrane-dense plaques of smooth muscle. The MAb 30B6 antigen is expressed in a wide number of chicken cell types (particularly smooth muscle cells, platelets, and endothelial cells), but not in erythrocytes. Some of the molecular characteristics of the MAb 30B6 antigen have been determined from immunoblotting, immunoaffinity chromatography, immunoprecipitation, cell extraction, and charge shift electrophoresis experiments. It is an integral sialoglycoprotein with an apparent molecular mass of 130 kD (reduced form)/107 kD (nonreduced form) in SDS PAGE. Another prominent glycoprotein species with an apparent molecular mass of 175 kD (reduced form)/165 kD (nonreduced form) in SDS PAGE is co-isolated on MAb 30B6 affinity columns, but appears to be antigenically distinct since it is not recognized by MAb 30B6 in immunoblotting or immunoprecipitation experiments. By virtue of its surface distributions relative to actin microfilaments and its integral protein character, we propose that the MAb 30B6 antigen is an excellent candidate for the function of directly or indirectly anchoring microfilaments to the membrane.

scholarly journals The neuropeptide proctolin induces phosphorylation of a 30 kDa protein associated with the thin filament in crustacean muscle

2001 ◽  
Vol 204 (15) ◽  
pp. 2627-2635 ◽  
Author(s):  
Berit Brüstle ◽  
Sabine Kreissl ◽  
Donald L. Mykles ◽  
Werner Rathmayer
Keyword(s):  
Molecular Mass ◽  
Thin Filament ◽  
Troponin I ◽  
Extensor Muscle ◽  
Apparent Molecular Mass ◽  
Muscle Fibres ◽  
Single Pair ◽  
Myofibrillar Proteins ◽  
Single Muscle ◽  
Western Blots

SUMMARYIn the isopod Idotea emarginata, the neuropeptide proctolin is contained in a single pair of motoneurones located in pereion ganglion 4. The two neurones supply dorsal extensor muscle fibres of all segments. Proctolin (1μmoll−1) potentiates the amplitude of contractures of single extensor muscle fibres elicited by 10mmoll−1 caffeine. In western blots of myofibrillar proteins isolated from single muscle fibres and treated with an anti-phosphoserine antibody, a protein with an apparent molecular mass of 30kDa was consistently found. The phosphorylation of this protein was significantly increased by treating the fibres with proctolin. After separation of myofibrillar filaments, a 30kDa protein was found only in the thin filament fraction. This protein is phosphorylated and detected by an antiserum against crustacean troponin I.

scholarly journals Role of calcium ions in the regulation of intramitochondrial metabolism. Properties of the Ca2+-sensitive dehydrogenases within intact uncoupled mitochondria from the white and brown adipose tissue of the rat

1980 ◽  
Vol 190 (1) ◽  
pp. 95-105 ◽  
Author(s):  
J G McCormack ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Epididymal Adipose Tissue ◽  
Ionophore A23187 ◽  
Mitochondrial Inner Membrane ◽  
Brown Adipose ◽  
Oxoglutarate Dehydrogenase

1. Increasing concentrations of both Ca2+ and Sr2+ (generated by using EGTA buffers) resulted in 4-fold increases in the initial activity of pyruvate dehydrogenase within intact uncoupled mitochondria from rat epididymal adipose tissue incubated in the presence of the ionophore A23187, ATP, Mg2+ and oligomycin. The k0.5 values (concentrations required for half-maximal effects) for Ca2+ and Sr2+ were 0.54 and 7.1 microM respectively. In extracts of the mitochondria, pyruvate dehydrogenase phosphate phosphatase activity was stimulated about 4-fold by Ca2+ and Sr2+, with k0.5 values of 1.08 and 6.4 microM respectively. 2. NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase appeared to be rate-limiting in the oxidation of threo-Ds-isocitrate and oxoglutarate by uncoupled mitochondria from brown adipose tissue of cold-adapted rats. Ca2+ (and Sr2+) diminished the Km for the oxidation of both threo-Ds-isocitrate and oxoglutarate. The kinetic constants for these oxidations were very similar to those obtained for the activities of NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of the mitochondria. In particular, the k0.5 values for Ca2+ were all in the range 0.2–1.6 microM and Sr2+ was found to mimic Ca2+, but with k0.5 values about 10 times greater. 3. Overall, the results of this study demonstrate that the activities of pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase may all be increased by Ca2+ and Sr2+ within intact mitochondria. In all cases the k0.5 values are close to 1 and 10 microM respectively, as found for the separated enzymes. Experiments on brown-adipose-tissue mitochondria incubated in the presence of albumin suggest that it may be possible to use the sensitivity of the dehydrogenases to Ca2+ as a means of assessing the distribution of Ca2+ across the mitochondrial inner membrane.

Increased sympathetic activity in rat white adipose tissue during prolonged fasting

1997 ◽  
Vol 272 (2) ◽  
pp. R656-R661 ◽  
Author(s):  
R. H. Migliorini ◽  
M. A. Garofalo ◽  
I. C. Kettelhut
Keyword(s):  
Adipose Tissue ◽  
White Adipose Tissue ◽  
Sympathetic Activity ◽  
Epididymal Adipose Tissue ◽  
Turnover Rates ◽  
Prolonged Fasting ◽  
Interscapular Brown Adipose Tissue ◽  
Brown Adipose ◽  
6 Hydroxydopamine ◽  
Fasted Rats

The effect of prolonged fasting on sympathetic activity was examined in rat white adipose tissue (WAT) and, for comparison purposes, in interscapular brown adipose tissue (IBAT). Preliminary experiments showed that 6-hydroxydopamine or tyramine administration to fed animals produced similar reductions in norepinephrine (NE) content of WAT and IBAT. Fasting for 48 h did not affect tissue NE content significantly, but induced a threefold increase in [3H]NE uptake by retroperitoneal and epididymal adipose tissue, contrasting with a 50% reduction in IBAT. Measured with DL-alpha-methyl-p-tyrosine, NE fractional rates of turnover were faster and calculated turnover rates were three times higher in retroperitoneal and epididymal tissue from fasted rats than in tissues from fed controls. In experiments with [3H]NE, although fractional rates did not change significantly, calculated NE turnover also increased in retroperitoneal and epididymal tissue after food deprivation. In contrast, in IBAT, NE turnover either did not change (measured with DL-alpha-methyl-p-tyrosine) or, in the experiments with [3H]NE, decreased significantly after fasting. These and other data suggest that a centrally controlled selective activation of WAT sympathetic fibers contributes to fasting lipolysis.

scholarly journals Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization

1994 ◽  
Vol 304 (3) ◽  
pp. 693-698 ◽  
Author(s):  
L W J Klomp ◽  
L van Rens ◽  
G J Strous
Keyword(s):  
Molecular Mass ◽  
Mechanical Damage ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
Apparent Molecular Mass ◽  
Gastric Mucin ◽  
Gastric Mucus ◽  
Apical Side ◽  
Sds Page ◽  
Glycosylation Inhibitor

Gastric mucin plays an important role in the protection of the stomach wall from chemical, microbiological and mechanical damage. We have previously isolated human gastric mucus glycoproteins and raised a polyclonal antiserum against these macromolecules. This antiserum specifically reacted with gastric mucins in immunoblotting experiments and stained mucous granules at the apical side of gastric surface epithelial cells. A similar staining pattern was obtained after incubation with an antiserum against rat gastric mucin. Next we used the antiserum in pulse-chase experiments of human stomach tissue explants. After short labelling periods with [35S]methionine and [35S]cysteine, the antiserum reacted with a polypeptide with an apparent molecular mass of approx. 500 kDa as determined by SDS/PAGE, which was converted after 90 min into a heterogeneous high-molecular-mass glycoprotein. This high-molecular-mass form, but not the 500 kDa polypeptide, was detectable in the culture medium after 2 h. This strongly suggests that the 500 kDa polypeptide is the precursor of the purified gastric mucin. Analysis of pulse-chase experiments by non-reducing SDS/PAGE revealed that the precursors form disulphide-linked oligomers early in biosynthesis, before the addition of O-linked sugars. After preincubation with the N-glycosylation inhibitor, tunicamycin, the apparent molecular mass of the precursor decreased marginally but consistently, indicating that N-linked glycan chains are present on the mucin precursor.

scholarly journals Effect of nutrition on subcellular localization of rat fat-cell lipoprotein lipase

1978 ◽  
Vol 172 (2) ◽  
pp. 239-245 ◽  
Author(s):  
A Vanhove ◽  
C Wolf ◽  
M Breton ◽  
M C Glangeaud
Keyword(s):  
Adipose Tissue ◽  
Lipoprotein Lipase ◽  
Plasma Membranes ◽  
Total Activity ◽  
Normal Diet ◽  
Epididymal Adipose Tissue ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Fat Cell ◽  
Intact Tissue

This study supports the possibility for multiple subcellular forms of lipoprotein lipase. 1. The total activity of lipoprotein lipase per g of intact epididymal adipose tissue from fed rats is much higher than that from starved rats. 2. The isolated fat-cells of fed and of starved rats have lipoprotein lipase of almost the same activity per g of fat-pads. The isolated fat-cells of starved rats have a much higher proportion of total activity per g of the intact tissue than do those of fed rats. 3. Under the conditions of homogenization used, only a small proportion of the total activity per g of intact tissue from fed rats was associated with the fat layer which floated to the top of the homogenate during low-speed centrifugation. The different proportions of the specific enzyme activity found in each subcellular fraction are described. 4. Lipoprotein lipase from plasma membranes and microsomal fractions from starved and fed rats was purified by affinity chromatography. 5. The total activity of microsomal lipoprotein lipase per g of intact adipose tissue is enhanced by a normal diet. 6. In intact epididymal adipose tissue from fed rats, the activity per g of tissue of lipoprotein lipase of plasma membranes is much higher than that in the same fraction from starved rats. By contrast, the activities per g of tissue in plasma membranes obtained from starved or from fed rats by collagenase treatment were similar.

Normal and Neoplastic Cells of Brown Adipose Tissue Express the Adhesion Molecule CD31

2006 ◽  
Vol 130 (4) ◽  
pp. 480-482 ◽  
Author(s):  
Renato Rosso ◽  
Marco Lucioni
Keyword(s):  
Adipose Tissue ◽  
Adhesion Molecule ◽  
Myxoid Liposarcoma ◽  
Cell Types ◽  
Brown Fat ◽  
Fat Cells ◽  
Neoplastic Cells ◽  
Brown Adipose ◽  
Formalin Fixed Paraffin Embedded ◽  
Well Differentiated

Abstract Context.—CD31 (platelet-endothelial cell adhesion molecule-1; PECAM-1), an adhesion molecule involved in the process of angiogenesis, is used as a marker of normal and neoplastic vascularization. During the assessment of angiogenesis and vascular invasion in a thymic carcinoid tumor, we observed unexpected immunostaining for CD31 in perithymic brown fat nests. Objective.—To determine whether CD31 is expressed by normal and neoplastic cells of brown fat, a tissue whose thermogenetic activity depends heavily on high perfusion. Design.—Formalin-fixed, paraffin-embedded archival tissues were immunostained by the labeled avidin-biotin method using antibodies against CD31 (clones JC70A and 1A10) after retrieval of heat-induced epitopes. Archival tissues included perithymic, periadrenal, axillary, and neck adipose tissue in which were embedded nests of brown fat (n = 15), hibernoma (n = 3), lipoma (n = 6), well-differentiated liposarcoma (n = 4), and myxoid liposarcoma (n = 4). Results.—Invariably, multivacuolated and univacuolated adipocytes of normal brown fat and hibernomas were intensely positive for the CD31 antigen. The immunostaining “decorated” cell membranes and the membranes of intracytoplasmic vacuoles. No expression of CD31 was found in normal adipocytes of white fat, in neoplastic cells of lipomas, or in multivacuolated lipoblasts of well-differentiated and myxoid liposarcomas. Conclusions.—The spectrum of cell types that express CD31 is expanded to include normal and neoplastic brown fat cells. We speculate that the expression of CD31 may play a role in the development and maintenance of the vascular network characterizing this specialized adipose tissue. Moreover, CD31 may inhibit the Bax-mediated apoptosis of brown fat cells. For practical purposes, CD31 may be used as an immunohistochemical marker for distinguishing between white and brown fat and for diagnosing hibernoma in paraffin sections.

Rutin synthase in fava d’anta: purification and influence of stressors

2009 ◽  
Vol 89 (5) ◽  
pp. 895-902 ◽  
Author(s):  
N Lucci ◽  
P Mazzafera
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Reaction Temperature ◽  
Enzyme Activities ◽  
Apparent Molecular Mass ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Intermediary Metabolite ◽  
Sds Page ◽  
Optimum Reaction

The flavonoid rutin is synthesized in plants from quercetin, via a process in which isoquercitrin is an intermediary metabolite. In this work, the activities of isoquercitrin synthase and rutin synthase, and the quercetin, isoquercitrin and rutin contents of fava d’anta plants stressed for water (drought and flooding) and salt (NaCl) were studied. In general, stress increased the contents of the three compounds and both enzyme activities. Semi-purified rutin synthase and isoquercitrin synthase showed Km values of 1.816 and 2.10 µM, respectively, with optimum reaction pHs of 5 and 7, respectively, and an optimum reaction temperature of 35°C. Rutin synthase was purified from leaf buds and showed an apparent molecular mass of 39 KDa by SDS-PAGE. Mass spectrometry analysis of the purified protein did not reveal any similarity to the few known sequenced glycosyltransferases.Key words: Dimorphandra mollis, faveiro, flavonoid, isoquercitrin, quercetin.

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