scholarly journals Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment

1990 ◽  
Vol 272 (3) ◽  
pp. 703-712 ◽  
Author(s):  
F Authier ◽  
M Janicot ◽  
F Lederer ◽  
B Desbuquois
Keyword(s):  
Acid Phosphatase ◽  
Plasma Membranes ◽  
Degradation Products ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Chloroquine Treatment ◽  
Endosomal Acidification ◽  
Rapid Generation

The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

Enhancing Effect of Surfactant and Protein on Hydrolysis of Thymolphthalein Monophosphate by Purified Prostatic Acid Phosphatase

1975 ◽  
Vol 21 (12) ◽  
pp. 1761-1765 ◽  
Author(s):  
Andras G Foti ◽  
Harvey Herschman ◽  
J Fenimore Cooper ◽  
Hedi imFeld
Keyword(s):  
Acid Phosphatase ◽  
Serum Proteins ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Prostatic Acid Phosphatase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Brij 35 ◽  
Hydrolysis Of ◽  
Protein Bovine Serum Albumin

Abstract Purified prostatic acid phosphatase catalyzes the hydrolysis of thymolphthalein monophosphate 10-fold faster if an optimal concentration of Brij 35 (a wetting agent) or protein (bovine serum albumin or human serum proteins) is present. Results of gel filtration, dialysis, and sucrose density-gradient centrifugation analysis suggest that the substrate must combine with detergent or protein before the enzyme can catalyze its hydrolysis.

Iron availability from meat

1978 ◽  
Vol 39 (3) ◽  
pp. 631-638 ◽  
Author(s):  
T. Hazell ◽  
D. A. Ledward ◽  
R. J. Neale
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Gel Filtration ◽  
Rat Muscle ◽  
Muscle Extract ◽  
Freeze Dried ◽  
Absorption Studies ◽  
Fe Complexes

1. The distribution of radioactive iron in59Fe-labelled rat muscle extract was determined using ge filtration. This showed that most (approximately 70%) of the radioactivity was associated with the heamatin compounds; myoglobin and haemoglobin.2. Raw beef and freeze-dried rat muscle were digested in vitro, under simulated physiological conditions, and after centrifugation the supernatants fractionated by gel filtration. The soluble products were haematin Fe complexes of molecular weight above 10 000 and non-haematin Fe compounds of molecular weight below 6000, the major products being the non-haematin Fe complexes. The soluble compounds were also separated by dialysis and, in rat muscle, it was found that the low-molecular-weight non-haematin compounds accounted for more than 80% of the total soluble iron.3. In vivo absorption studies with rats showed the Fe in a digested muscle dialysate to be more readily absorbed than that from an aqueous muscle extract which itself was more readily absorbed than the Fe from whole blood.4. It may not, therefore, be the haemoproteins per se which are responsible for the high availability of Fe in meat, but rather the nature of their degradation products, formed by digestion within the meat environment.

Purification of bovine lens cell-to-cell channels composed of connexin44 and connexin50

1995 ◽  
Vol 108 (9) ◽  
pp. 3091-3098 ◽  
Author(s):  
N. Konig ◽  
G.A. Zampighi
Keyword(s):  
Alkaline Phosphatase ◽  
Column Chromatography ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Sds Page ◽  
Lens Cell ◽  
Fetal Bovine ◽  
Complete Cell ◽  
Nonionic Detergents

Cell-to-cell channels composed of connexin44 and connexin50 were purified from plasma membranes of calf and fetal bovine lenses. The channels were treated with the nonionic detergents octyl-beta-D-glucopyranoside and decyl-beta-D-maltopyranoside, and the channel/detergent complexes purified by ion and gel filtration column chromatography. In negative staining, the channels appeared as annuli 11 +/- 0.6 nm (s.d., n = 105) in diameter and as 16 +/- 0.8 nm (s.d., n = 96) long particles which corresponded to top and side views of ‘complete’ cell-to-cell channels. The purified cell-to-cell channels were composed principally of a protein, called MP70, that appeared as a diffuse 55–75 kDa band in SDS-PAGE. Dephosphorylation with alkaline phosphatase transformed the diffuse 55–75 kDa band into two distinct bands of almost equal intensity. Immunoblotting showed the bands to be connexin44 and connexin50, respectively. The antibodies also recognized weaker bands composed of the unphosphorylated form of both connexins. The connexins appear to be processed independently ‘in vivo’. The unphosphorylated form of connexin50 was present in channels and membranes from fetal, calf and adult bovine lenses, while unphosphorylated connexin44 only in channels purified from fetal lenses. Therefore, lens cell-to-cell channels are composed principally of equal amounts of phosphorylated connexins 44 and 50 that appear to be assembled in the same channel (‘hybrid’).

scholarly journals Degradation of glucagon in isolated liver endosomes. ATP-dependence and partial characterization of degradation products

1991 ◽  
Vol 280 (1) ◽  
pp. 211-218 ◽  
Author(s):  
F Authier ◽  
B Desbuquois
Keyword(s):  
Degradation Products ◽  
Glucagon Receptor ◽  
Free System ◽  
Chloroquine Treatment ◽  
Endosomal Acidification ◽  
A Cell ◽  
Poly Ethylene ◽  
Gradient Fractionation

Endosomes have recently been identified as one major site of glucagon degradation in intact rat liver. In this study, a cell-free system has been used to assess the role of ATP-dependent acidification in endosomal glucagon degradation and identify the glucagon products generated. Percoll gradient fractionation of Golgi-endosomal fractions prepared 10-30 min after injection of [125I]iodoglucagon showed a time-dependent shift of the radioactivity towards high densities. Regardless of time, the radioactivity was less precipitable by trichloroacetic acid (Cl3Ac) at high densities than at low densities. Chloroquine treatment slightly increased the density shift of the radioactivity and decreased its Cl3Ac-precipitability throughout the gradient. Incubation of endosomal fractions containing [125I]iodoglucagon in 0.15 M-KCl at 30 degrees C resulted in a time- and pH-dependent generation of Cl3Ac-soluble radioactivity, with a maximum at pH 4 (t1/2, 7 min). At pH 5, 1,10-phenanthroline, bacitracin and p-chloromercuribenzoic acid partially inhibited [125I]iodoglucagon degradation. At pH 6-7, ATP stimulated [125I]iodoglucagon degradation by 5-10-fold and caused endosomal acidification as judged from Acridine Orange uptake. The effects of ATP were inhibited by chloroquine, monensin, N-ethylmaleimide and dansylcadaverine. Poly(ethylene glycol) (PEG) precipitation of the radioactivity associated with endosomes showed that lowering the pH below 5.5 caused dissociation of the glucagon-receptor complex, and that, regardless of incubation conditions, all degraded [125I]iodoglucagon diffused extraluminally. On h.p.l.c., at least three products less hydrophobic than [125I]iodoglucagon were identified in incubation mixtures along with monoiodotyrosine. Radiosequence analysis of the products revealed one major cleavage located C-terminally to Tyr-13 and two minor cleavages affecting Thr-5-Phe-6 and Phe-6-Thr-7 bonds. It is concluded that glucagon degradation in liver endosomes is functionally linked to ATP-dependent endosomal acidification and involves several cleavages in the glucagon sequence.

Renal metabolism of calcitonin

1988 ◽  
Vol 254 (4) ◽  
pp. F593-F600 ◽  
Author(s):  
R. E. Simmons ◽  
J. T. Hjelle ◽  
C. Mahoney ◽  
L. J. Deftos ◽  
W. Lisker ◽  
...  
Keyword(s):  
Brush Border ◽  
Degradation Products ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Human Calcitonin ◽  
Renal Handling ◽  
Renal Metabolism ◽  
Brush Border Enzyme

The kidneys account for approximately two-thirds of the metabolism of calcitonin, but relatively little is known regarding the details thereof. To further characterize this process, we examined the renal handling and metabolism of human calcitonin (hCT) by the isolated perfused rat kidney. We also studied the degradation of radiolabeled salmon calcitonin (sCT) by subcellular fractions prepared from isolated rabbit proximal tubules. The total renal (organ) clearance of immunoreactive hCT by the isolated kidney was 1.96 +/- 0.18 ml/min. This was independent of the perfusate total calcium concentration from 5.5 to 10.2 mg/dl. Total renal clearance exceeded the glomerular filtration rate (GFR, 0.68 +/- 0.05 ml/min), indicating filtration-independent removal. Urinary calcitonin clearance as a fraction of GFR averaged 2.6%. Gel filtration chromatography of medium from isolated kidneys perfused with 125I-labeled sCT showed the principal degradation products to be low molecular weight forms eluting with monoiodotyrosine. Intermediate size products were not detected. In the subcellular fractionation experiments, when carried out at pH 5.0, calcitonin hydrolysis exclusively followed the activities of the lysosomal enzyme N-acetyl-beta-glucosaminidase. Typically, at pH 7.5, 42% of total degradation occurred in the region of the brush-border enzyme alanyl aminopeptidase and 29% occurred in the region of the cytosolic enzyme phosphoglucomutase. Although 9% of the calcitonin-degrading activity was associated with basolateral membrane fractions, most of this activity could be accounted for by the presence of brush-border membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Oestrogen receptor of mammary gland. Inhibition of aggregation and characterization of receptor from lactating gland in the presence of sodium bromide

1978 ◽  
Vol 169 (3) ◽  
pp. 481-488 ◽  
Author(s):  
Ferdinando Auricchio ◽  
Andrea Rotondi ◽  
Ettore Schiavone ◽  
Francesco Bresciani
Keyword(s):  
Oestrogen Receptor ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Complete Disappearance ◽  
Number Of Binding Sites ◽  
Stokes Radius

1. When NaBr, a chaotropic salt, is added, in concentrations ranging from 0.5m to 2m, to low-salt mammary cytosol, (i) age-dependent aggregation of oestrogen receptor is inhibited, (ii) the receptor sediments as a sharp peak at 4.2S on sucrose-gradient centrifugation, with complete disappearance of heavier forms, and (iii) on gel filtration with Sephadex G-200, the receptor is included in the gel matrix. On a calibrated column, the receptor has a Stokes radius of 3.7nm (±6%). 2. Because NaBr inhibits interaction of receptor with other components of cytosol, the values of the sedimentation coefficient, measured by sucrose-gradient sedimentation, and of the Stokes radius, measured by gel filtration, can be accepted with confidence. From these values, it can be computed that the oestrogen-receptor form in NaBr has a mol.wt. of 64000, with a frictional ratio of 1.4. 3. Also, inhibition of aggregation by NaBr allows a 30–90-fold purification of oestrogen receptor. Analysis of this partially purified receptor by sucrose-gradient sedimentation and gel filtration in NaBr gives the same results as for receptor in crude cytosol. On electrofocusing on a pH5–8 gradient, the partially purified oestrogen receptor focuses at pH6.2. On removal of NaBr, receptor aggregates even in this partially purified state. It seems likely that at the protein and ionic concentrations of cytoplasm in vivo, the 64000-mol.wt. receptor form is part of higher states of self- and/or hetero-association with other cytoplasmic components. 4. NaBr up to a concentration of 2m does not inhibit binding of oestrogen by receptor, nor does it decrease the affinity of the interaction (KD≃8.9×10−10m). The total number of binding sites in cytosol, however, decreases by approx. 10%, but this decrease may actually be the result of elimination of lower-affinity binding by non-receptor components of cytosol. 5. NaSCN, another chaotropic salt, was also tested but gave less satisfactory results with the mammary cytosol than with uterine cytosol. EDTA was omitted from the buffers because it favours aggregation of mammary oestrogen receptor. KCl (0.4m), sucrose (15%) and ZnSO4 (3mm) did not prevent aggregation of receptor.

scholarly journals Isolation and partial characterization of an acid phosphatase from Artemisia vulgaris pollen extract

2002 ◽  
Vol 67 (8-9) ◽  
pp. 567-572 ◽  
Author(s):  
Tanja Cirkovic-Velickovic ◽  
Marija Gavrovic-Jankulovic ◽  
Mirjana Bukilica ◽  
Ljuba Mandic ◽  
Spomenka Petrovic ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Gel Filtration ◽  
Sulfhydryl Group ◽  
Inhibitory Effect ◽  
Tween 20 ◽  
Pollen Extract ◽  
Gel Chromatography ◽  
Artemisia Vulgaris ◽  
Nitrophenyl Phosphate

An acid phosphatase from an extract of mugwort (Artemisia vulgaris) pollen was purified by a factor of 48 by a combination of ion exchange and gel-chromatography. The molecular weights of the enzyme were 76 kDa and 73 kDa, determined by gel filtration on a Sephadex G-100 sf column and by SDS PAGE(under reducing and non-reducing conditions), respectively. In analytical isoelectrofocusing, the enzyme appears as two very close bands pI at about 4.2. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.16mM. The purified enzyme has broad specificity, and hydrolyses p-nitrophenyl phosphate and ?-naphthyl phosphate. Pyrophosphate and O-phospho-L-tyrosine were estimated to be the best substrates for this enzyme as potential in vivo substrates. The enzyme is inhibited competitively by phosphate (Ki = 1.25 mM), molybdate (Ki = 0.055 mM) and pyrophosphate (Ki = 6.7 mM) and non-competitively by fluoride (Ki = 9.8 mM). Metal ions such as Hg2+, Cu2+ and Zn2+ express an inhibitory effect on the enzyme, while the enzyme is slightly activated by non-ionic detergents, Tween 20 and Triton X-100. There is no change in the enzyme activity in the presence of tartrate, citrate, EDTA, 1,10-phenanthroline and sulfhydryl-group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.

scholarly journals Membrane protein phosphotyrosine phosphatase in rabbit kidney. Proteolysis activates the enzyme and generates soluble catalytic fragments

1987 ◽  
Vol 243 (3) ◽  
pp. 747-754 ◽  
Author(s):  
S A Rotenberg ◽  
D L Brautigan
Keyword(s):  
Phosphatase Activity ◽  
Plasma Membranes ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Cell Types ◽  
Total Activity ◽  
Rabbit Kidney ◽  
Ph Optimum ◽  
Fresh Tissue

Most protein phosphotyrosine phosphatases (PPT-phosphatases) have been recovered from the cytosol of various cell types and tissues. The present study explores the properties of PPT-phosphatases in rabbit kidney membranes prepared by centrifugation at 100,000 g. More of the total activity was recovered in membranes from fresh (45%) compared with frozen-and-thawed (36%) tissue. However, extracts of fresh tissue had only 15-30% as much total PPT-phosphatase activity. Up to 3-fold activation of cytosolic and membrane PPT-phosphatases occurred during preparation, an effect most evident when fresh tissue was homogenized in buffers containing multiple proteinase inhibitors. These inhibitors apparently block some, but not all, digestion of proteins that mask PPT-phosphatase activity. Incubation of membranes prepared from fresh tissue with added trypsin, papain or thermolysin in each case caused activation of PPT-phosphatase as well as generation of a soluble catalytic fragment. The fragment also was generated by the action of endogenous proteinases during repeated centrifugation and was isolated from these supernatants by DEAE-Sepharose, Zn2+-affinity and gel-filtration chromatography. The fragment had Mr approx. 33,000, had a neutral pH optimum, was inhibited by 50% by 100 microM-vanadate, and was insensitive to the alkaline-phosphatase inhibitors EDTA and levamisole. Although the chromatographic behaviour and lability of the fragment were distinct from those of the predominant cytosolic PPT-phosphatase, some cytosolic PPT-phosphatases exhibited properties consistent with the suggestion that they are fragments derived by proteolysis of PPT-phosphatases in membranes. Localization of PPT-phosphatases in plasma membranes would facilitate reaction with receptor/kinases in vivo.

scholarly journals Galectin-4 and Small Intestinal Brush Border Enzymes Form Clusters

1997 ◽  
Vol 8 (11) ◽  
pp. 2241-2251 ◽  
Author(s):  
E. Michael Danielsen ◽  
Bo van Deurs
Keyword(s):  
Brush Border ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Aminopeptidase N ◽  
Intestinal Lumen ◽  
Immunogold Electron Microscopy ◽  
Brush Border Enzymes ◽  
Intestinal Brush Border ◽  
Mucosal Explants

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly “trapped” by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

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