scholarly journals Proteolytic cleavage of haptoglobin occurs in a subcompartment of the endoplasmic reticulum: evidence from membrane fusion in vitro.

1993 ◽  
Vol 123 (2) ◽  
pp. 285-291 ◽  
Author(s):  
M Wassler ◽  
E Fries
Keyword(s):  
Liver Homogenate ◽  
Rat Hepatocytes ◽  
Translation Product ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Gtp Hydrolysis ◽  
Cytoplasmic Proteins ◽  
Gamma S ◽  
Stimulation Of

The primary translation product of haptoglobin mRNA is a 45-kD polypeptide which is proteolytically cleaved shortly after its synthesis. Previous studies have indicated that the cleavage of this proform of haptoglobin occurs in the ER. In an attempt to characterize the cleaving enzyme, we found that upon incubation of microsomes from rat hepatocytes pulse labeled with [35S]methionine, little cleavage of labeled prohaptoglobin occurred. In contrast, when cells whose cytoplasmic proteins had been released by saponin treatment were incubated, 30-40% of the prohaptoglobin was cleaved. The addition of GTP caused a twofold stimulation, which was abolished by the nonhydrolyzable analog GTP gamma S. With a homogenate of the cells, the addition of GTP resulted in a fourfold stimulation of the degree of cleavage--from 15 to 60%. Differential centrifugation revealed that most of the cleaving activity resided in membranes sedimenting similarly to mitochondria and to a small fraction of the ER. These rapidly sedimenting membranes were therefore prepared from a rat liver homogenate. Upon treatment with high salt, light membranes were released which, when incubated with microsomes of pulse-labeled hepatocytes in the presence of detergent (and in the absence of GTP), induced specific cleavage of prohaptoglobin. The cleaving enzyme had an alkaline pH optimum indicating that it was not of lysosomal origin. These results suggest that cleavage of prohaptoglobin occurs in a subcompartment of the ER. Apparently, the connection between this compartment and the bulk of the ER is broken upon saponin treatment or homogenization but can be reestablished through a process requiring GTP hydrolysis.

COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargo

2000 ◽  
Vol 113 (1) ◽  
pp. 135-144 ◽  
Author(s):  
R. Pepperkok ◽  
J.A. Whitney ◽  
M. Gomez ◽  
T.E. Kreis
Keyword(s):  
Protein Transport ◽  
Ectopic Expression ◽  
Small Gtpase ◽  
Coated Vesicles ◽  
Gtp Hydrolysis ◽  
Rapid Accumulation ◽  
Gamma S

Microinjection of the slowly hydrolyzable GTP analogue GTP(gamma)S or the ectopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1[Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15 degrees C and by microinjection of antibodies against (beta)-COP. Anterograde and retrograde membrane protein transport markers, which have been previously shown to be incorporated into COPI vesicles between the endoplasmic reticulum and Golgi complex, are depleted from the GTP(gamma)S or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-localize with these markers. These in vivo data corroborate recent in vitro work, suggesting that GTP(gamma)S and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of cargo into COPI coated vesicles and buds.

scholarly journals Increased oxidation of uroporphyrinogen by an inducible liver microsomal system. Possible relevance to drug-induced uroporphyria

1988 ◽  
Vol 250 (1) ◽  
pp. 161-169 ◽  
Author(s):  
F De Matteis ◽  
C Harvey ◽  
C Reed ◽  
R Hempenius
Keyword(s):  
Chick Embryo ◽  
Drug Metabolism ◽  
Microsomal Fraction ◽  
Marked Decrease ◽  
Rat Hepatocytes ◽  
Drug Induced ◽  
Dependent Mechanism ◽  
Stimulation Of

1. The hypothesis that uroporphyria-inducing drugs stimulate the oxidation of uroporphyrinogen by a microsomal NADPH-dependent mechanism was tested. 2. 3,4,3′,4′-Tetrachlorobiphenyl, a very effective inducer of uroporphyria in chick-embryo hepatocyte cultures, stimulates the NADPH-dependent oxidation of uroporphyrinogen by chick-embryo microsomal fraction in vitro. 3. Two different actions of 3,4,3′,4′-tetrachlorobiphenyl are apparently required for this effect: (a) induction of a microsomal system by treatment in vivo and (b) interaction with the induced microsomal fraction in vitro, producing an oxidizing species. 4. The analogue 2,4,2′,4′-tetrachlorobiphenyl is relatively ineffective in both the production of porphyria in culture and the stimulation of porphyrinogen oxidation in vitro. 5. Rat hepatocytes do not develop uroporphyria when treated with polychlorinated biphenyls in culture, yet they respond to these drugs with typical induction of cytochrome P-448-dependent drug metabolism. 6. These data provide support for the hypothesis of an increased oxidation of uroporphyrinogen in drug-induced uroporphyria, but also suggest that induction of cytochrome P-448 is not the only factor involved. 7. Both I and III isomers of uroporphyrin and heptacarboxylate porphyrin accumulate when chicken hepatocytes are made uroporphyric by drugs; treatment with desferrioxamine causes a marked decrease in both isomers, suggesting that iron may be involved in the accumulation of both.

scholarly journals Stimulation of [1-14C]oleate oxidation to 14CO2 in isolated rat hepatocytes by the catecholamines, vasopressin and angiotensin. A possible mechanism of action

1983 ◽  
Vol 212 (1) ◽  
pp. 85-91 ◽  
Author(s):  
M C Sugden ◽  
D I Watts
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Tricarboxylic Acid Cycle ◽  
Rat Hepatocytes ◽  
Tricarboxylic Acid ◽  
Isolated Rat Hepatocytes ◽  
Acid Cycle ◽  
14Co2 Production ◽  
Oxoglutarate Dehydrogenase ◽  
Stimulation Of

Adrenaline, noradrenaline, vasopressin and angiotensin increased 14CO2 production from [1-14C]oleate by hepatocytes from fed rats but not by hepatocytes from starved rats. The hormones did not increase 14CO2 production when hepatocytes from fed rats were depleted of glycogen in vitro. Increased 14CO2 production from]1-14C]oleate in response to the hormones was observed when hepatocytes from starved rats were incubated with 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase. 3-Mercaptopicolinate inhibited uptake and esterification of [1-14C]oleate, slightly increased 14CO2 production from [1-14C]oleate and greatly increased the [3-hydroxybutyrate]/[acetoacetate] ratio. In the presence of 3-mercaptopicolinate 14CO2 production in response to the catecholamines was blocked by the α-antagonist phentolamine and required extracellular Ca2+. The effects of vasopressin and angiotensin were also Ca2+-dependent. The actions of the hormones of 14CO2 production from [I-14C]oleate by hepatocytes from starved rats in the presence of 3-mercaptopicolinate thus have the characteristics of the response to the hormones found with hepatocytes from fed rats incubated without 3-mercaptopicolinate. The stimulatory effects of the hormones on 14CO2 production from [1-14C]oleate were not the result of decreased esterification (as the hormones increased esterification) or increased β-oxidation. It is suggested that the effect of the hormones to increase 14CO2 production from [1-14C]oleate are mediated by CA2+-activation of NAD+-linked isocitrate dehydrogenase, the 2-oxoglutarate dehydrogenase complex, and/or electron transport. The results also demonstrate that when the supply of oxaloacetate is limited it is utilized for gluconeogenesis rather than to maintain tricarboxylic acid-cycle flux.

scholarly journals Activation of protein kinase B β and γ isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase B α

1998 ◽  
Vol 331 (1) ◽  
pp. 299-308 ◽  
Author(s):  
Kay S. WALKER ◽  
Maria DEAK ◽  
Andrew PATERSON ◽  
Kevin HUDSON ◽  
Philip COHEN ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Rat Hepatocytes ◽  
Dependent Protein Kinase ◽  
L6 Myotubes ◽  
Dependent Protein ◽  
Stimulation Of

The regulatory and catalytic properties of the three mammalian isoforms of protein kinase B (PKB) have been compared. All three isoforms (PKBα, PKBβ and PKBγ) were phosphorylated at similar rates and activated to similar extents by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Phosphorylation and activation of each enzyme required the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2, as well as PDK1. The activation of PKBβ and PKBγ by PDK1 was accompanied by the phosphorylation of the residues equivalent to Thr308 in PKBα, namely Thr309 (PKBβ) and Thr305 (PKBγ). PKBγ which had been activated by PDK1 possessed a substrate specificity identical with that of PKBα and PKBβ towards a range of peptides. The activation of PKBγ and its phosphorylation at Thr305 was triggered by insulin-like growth factor-1 in 293 cells. Stimulation of rat adipocytes or rat hepatocytes with insulin induced the activation of PKBα and PKBβ with similar kinetics. After stimulation of adipocytes, the activity of PKBβ was twice that of PKBα, but in hepatocytes PKBα activity was four-fold higher than PKBβ. Insulin induced the activation of PKBα in rat skeletal muscle in vivo, with little activation of PKBβ. Insulin did not induce PKBγ activity in adipocytes, hepatocytes or skeletal muscle, but PKBγ was the major isoform activated by insulin in rat L6 myotubes (a skeletal-muscle cell line).

Regulation of acyl CoA: Cholesterol acyl transferase (ACAT) activity by mevalonate and cholesterol in isolated rat hepatocytes during perinatal development

1986 ◽  
Vol 6 (8) ◽  
pp. 735-740
Author(s):  
G. Bruscalupi ◽  
L. Conti Devirgiliis ◽  
S. Leoni ◽  
F. Piemonte ◽  
A. Trentalance
Keyword(s):  
Developmental Stages ◽  
Rat Hepatocytes ◽  
Differential Sensitivity ◽  
Substrate Effect ◽  
Acyl Transferase ◽  
Cholesterol Acyl Transferase ◽  
Isolated Rat Hepatocytes ◽  
Acat Activity ◽  
Stimulation Of

Acyl CoA: cholesterol acyl transferase (ACAT) activity presents marked oscillations and differential sensitivity to the “in vitro” stimulation of the kinase-phosphatase modulatory system in the perinatal rat liver. The regulation of this enzyme activity by some modulators generally active in adulthood, such as cholesterol, lipoproteins and mevalonate, has been studied in hepatocytes isolated at different developmental stages. A lack of effect of mevalonate and a positive effort of lipoprotein cholesterol have been observed at the fetal and neonatal stages. A differential prevalence is suggested of one of the two modulatory mechanisms (phosphorylation-dephosphorylation system, or substrate effect) at each developmental stage.

Incorporation of tubulin subunits into dimers requires GTP hydrolysis

1993 ◽  
Vol 106 (2) ◽  
pp. 627-632 ◽  
Author(s):  
A. Fontalba ◽  
R. Paciucci ◽  
J. Avila ◽  
J.C. Zabala
Keyword(s):  
Protein Folding ◽  
Atp Hydrolysis ◽  
In Vitro Translation ◽  
Magnesium Ions ◽  
Gtp Hydrolysis ◽  
Step Process ◽  
Tubulin Isotypes ◽  
Cytoplasmic Proteins ◽  
Formation Of Complexes

A toroid multisubunit complex of 800–900 kDa has been implicated in assisting protein folding of at least two cytoplasmic proteins, actin and tubulin. This process is dependent on the presence of magnesium ions and ATP hydrolysis. In vitro translation of cDNAs encoding different alpha- and beta-tubulin isotypes also gives rise to the formation of complexes of about 300 kDa. These complexes have been functionally implicated in the incorporation of tubulin monomers within the tubulin heterodimer. This work shows that, in addition to ATP hydrolysis, the incorporation of newly synthesized tubulin subunits into functional heterodimers requires GTP hydrolysis in the presence of magnesium ions. A two-step process is suggested, a first ATP-dependent step in which the 900 kDa complexes are implicated in a similar way to the step taking place in actin folding, and a second GTP-dependent step in which the 300 kDa complexes are involved in the assembly of the heterodimer.

Activation of Inactive Plasma Renin: Evidence that Both Cryoactivation and Acid-Activation Work by Liberating a Neutral Serine Protease from Endogenous Inhibitors

1978 ◽  
Vol 55 (s4) ◽  
pp. 135s-138s ◽  
Author(s):  
Steven A. Atlas ◽  
John H. Laragh ◽  
Jean E. Sealey
Keyword(s):  
Serine Protease ◽  
Plasma Renin ◽  
Lima Bean ◽  
Trypsin Inhibitors ◽  
Soya Bean ◽  
Acid Activation ◽  
Alkaline Ph ◽  
Two Phase ◽  
Ph Optimum

1. We have found that ‘acid’-activation of inactive human plasma renin is a two-phase process. About 30% of activation occurs during dialysis to pH 3·3; the remaining 70% occurs at alkaline pH. 2. The ‘alkaline phase’ of activation has a pH optimum between 7·5 and 8·4. It is inhibited by unacidified plasma and by soya-bean or lima-bean trypsin inhibitors. 3. ‘Cryoactivation’ of inactive plasma renin, which occurs at −4°C and alkaline pH, is also inhibited by soya-bean or lima-bean trypsin inhibitors and by the serine protease inhibitors di-isopropylphosphorofluoridate and benzamidine. 4. Thus endogenous neutral serine proteases participate in the activation of inactive plasma renin in vitro. Their action is prevented in the circulation by inhibitors which are inactivated by acid or cold.

Prostaglandins E2 and F2α are not involved in the monocytic-product (interleukin-1)-induced stimulation of hepatic fibrinogen synthesis in rats

1988 ◽  
Vol 74 (5) ◽  
pp. 477-483 ◽  
Author(s):  
J. C. W. M. Holtslag ◽  
H. J. Moshage ◽  
J. F. van Pelt ◽  
J. A. G. M. Kleuskens ◽  
F. W. J. Gribnau ◽  
...  
Keyword(s):  
Acute Phase ◽  
Acute Phase Response ◽  
Second Messengers ◽  
Interleukin 1 ◽  
Rat Hepatocytes ◽  
Phase Response ◽  
Mrna Content ◽  
Stimulation Of

1. Monocytic products, especially interleukin-1 (IL-1), play an important role in the acute-phase response. Prostaglandins have been shown to act as second messengers in several physiological alterations of the acute-phase response, such as fever, muscle wasting and immunoregulation. The present study was undertaken to determine the role of prostaglandins in the monocytic-product-induced stimulation of the hepatic synthesis of fibrinogen, a well-known acute-phase protein. 2. Prostaglandin (PG) E2, PGF2α and 16,16-dimethyl-PGE2 did not stimulate fibrinogen synthesis and fibrinogen polypeptide mRNA content when administered intraperitoneally to rats or when added to monolayer cultures of rat hepatocytes. 3. Cyclo-oxygenase inhibitors did not abolish the stimulation of fibrinogen synthesis and its mRNA content induced by monocytic products in vivo or in vitro. 4. These findings indicate that the enhanced synthesis of fibrinogen induced by monocytic products (including IL-1) during the acute-phase response is not mediated by prostaglandins or other products of the cyclo-oxygenase pathway of arachidonic acid.

scholarly journals Comparison in different species of biliary bilirubin-IX α conjugates with the activities of hepatic and renal bilirubin-IX α-uridine diphosphate glycosyltransferases

1977 ◽  
Vol 164 (3) ◽  
pp. 737-746 ◽  
Author(s):  
J Fevery ◽  
M Van de Vijver ◽  
R Michiels ◽  
K P M Heirwegh
Keyword(s):  
Glucuronic Acid ◽  
Mammalian Species ◽  
Liver Homogenate ◽  
Species Variation ◽  
Bile Pigment ◽  
Uridine Diphosphate ◽  
Ph Optimum ◽  
Equal Importance ◽  
Liver And Kidney

The bilrubin-IXalpha conjugates in bile and the activities of bilirubin-IX alphax–UDP-glycosyltransferases in liver and kidney were determined for ten species of mammals and for the chicken. 1. In the mammalian species, bilirubin-IX alpha glucuronide was the predominant bile pigment. Excretion of neutral glycosides was unimportant, except in the cat, the mouse, the rabbit and the dog, where glucose and xylose represented 12–41% of total conjugating groups bound to bilirubin-IX alpha. In chicken bile, glucoside and glucuronide conjugates were of equal importance. They probably represent only a small fraction of the total bile pigment. 2. The transferase activities in liver showed pronounced species variation. This was also apparent with regard to activation by digitonin, pH optimum and relative activities of transferases acting on either UDP-glucuronic acid or neutral UDP-sugars. 3. Man, the dog, the cat and the rat excrete bilirubin-IX alpha largely as diconjugated derivatives. In general, diconjugated bilirubin-IX alpha could also be synthesized in vitro with liver homogenate, bilirubin-IX alpha and UDP-sugar. In contrast, for the other species examined, bilirubin pigments consisted predominantly of monoconjugated bilirubin-IX alpha. Synthesis in vitro with UDP-glucuronic acid, UDP-glucose or UDP-xylose as the sugar donor led exclusively to the formation of monoconjugated bilirubin-IX alpha. 4. The transferase activities in the kidney were restricted to the cortex and were important only for the rat and the dog. No activity at all could be detected for several species, including man. 5. Comparison of the transferase activities in liver with reported values of the maximal rate of excretion in bile suggests a close linkage between conjugation and biliary secretion of bilirubin-IX alpha.

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