Phospholipid Transfer from ER to the Peribacteroid Membrane in Soybean Nodules

1985 ◽  
Vol 40 (1-2) ◽  
pp. 73-79 ◽  
Author(s):  
R. B. Mellor ◽  
T. M. I. E. Christensen ◽  
S. Bassarab ◽  
D. Werner
Keyword(s):  
Membrane Fraction ◽  
Adenosine Triphosphatase ◽  
Rhizobium Japonicum ◽  
Choline Kinase ◽  
Cytoplasmic Fraction ◽  
Peribacteroid Membrane ◽  
Choline Phosphotransferase ◽  
Membrane Atpase ◽  
Phospholipid Transfer

Organelle membranes subfractionated from nodules of Glycine max differed in lipid compo­sition. Of the enzymes assembling phospholipids, choline kinase (EC 2.7.1.32) was recovered in the cytoplasmic fraction whereas CDP choline 1,2-diacylglyceride choline phosphotransferase (EC2.7.8.2) was located in the ER. When methyl [14C]CDP choline was supplied to nodules in vivo radioactivity was found in the ER. On longer labelling periods [14C]phosphatidylcholine spread into denser regions of the gradient, including the peribacteroid membrane (which surrounds the Rhizobium japonicum symbiont). [14C]Phosphatidylcholine was a notable component of such membranes. Adenosine triphosphatase (EC 3.6.1.3) activity in the peribacteroid membrane fraction was compared to that of other major organelle fractions. The peribacteroid membrane ATPase was equally active on pH 6.0 and 8.0, was stimulated by K+ and inhibited by DES, DCCD and MoO4. In these respects it was more similar to the ATPase activity in the ER and mitochondria than in tonoplast or cytoplasm. The results are discussed in relation to modes of peribacteroid membrane biogenesis, in­cluding enzyme modification.

scholarly journals Dimeric heat shock protein 40 binds radial spokes for generating coupled power strokes and recovery strokes of 9 + 2 flagella

2008 ◽  
Vol 180 (2) ◽  
pp. 403-415 ◽  
Author(s):  
Chun Yang ◽  
Heather A. Owen ◽  
Pinfen Yang
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Adenosine Triphosphatase ◽  
Stable Conformation ◽  
Initiation And Propagation ◽  
Evolutionarily Conserved ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Hsp 40

T-shape radial spokes regulate flagellar beating. However, the precise function and molecular mechanism of these spokes remain unclear. Interestingly, Chlamydomonas reinhardtii flagella lacking a dimeric heat shock protein (HSP) 40 at the spokehead–spokestalk juncture appear normal in length and composition but twitch actively while cells jiggle without procession, resembling a central pair (CP) mutant. HSP40− cells begin swimming upon electroporation with recombinant HSP40. Surprisingly, the rescue doesn't require the signature DnaJ domain. Furthermore, the His-Pro-Asp tripeptide that is essential for stimulating HSP70 adenosine triphosphatase diverges in candidate orthologues, including human DnaJB13. Video microscopy reveals hesitance in bend initiation and propagation as well as irregular stalling and stroke switching despite fairly normal waveform. The in vivo evidence suggests that the evolutionarily conserved HSP40 specifically transforms multiple spoke proteins into stable conformation capable of mechanically coupling the CP with dynein motors. This enables 9 + 2 cilia and flagella to bend and switch to generate alternate power strokes and recovery strokes.

Digestion of tripeptides and disaccharides: relationship with brush border hydrolases

1976 ◽  
Vol 231 (1) ◽  
pp. 87-92 ◽  
Author(s):  
C Arvanitakis ◽  
J Ruhlen ◽  
J Folscroft ◽  
JB Rhodes
Keyword(s):  
Epithelial Cell ◽  
Intestinal Epithelial Cell ◽  
Intestinal Epithelial ◽  
Cytoplasmic Fraction ◽  
Perfusion Studies ◽  
Intestinal Digestion ◽  
Microvillus Membrane ◽  
Hydrolysis Of ◽  
Glycyl Glycine

Intestinal digestion of two tripeptides (leucyl-glycyl-glycine, prolyl-glycyl-glycine) and two disacchrarides (sucrose, maltose) was examined in the hamster by intestinal perfusion in vivo and hydrolysis of the substrates by microvillus membranes. Perfusion studies showed that luminal disappearance rates of leucyl-glycl-glycine were significantly higher than prolyl-glycyl-glycine (P less than o.001), sucrose (P less than 0.001), and maltose (P less than 0.005). Hydrolytic products of leucyl-glycyl-glycine, sucrose, and maltose were detected in the gut lumen in appreciable concentrations, whereas negligible concentrations of prolyl-glycyl-glycine products were present. Leucyl-glycyl-glycine hydrolysis in microvillus membranes was markedly higher than prolyl-glycyl-glycine (P less than 0.001), which was predominant in the cytoplasmic fraction. These results indicate that leucyl-glycyl-glycine, like sucrose and maltose, is hydrolyzed at the membrane. With some tripeptides, i.e., leucyl-glycyl-glycine, digestion occurs at the microvillus membrane with subsequent transport of hydrolytic products into the intestinal epithelial cell. Other tripeptides, i.e., prolyl-glycyl-glycine, may cross the membrane and undergo intracellular hydrolysis by cytoplasmic peptidases.

scholarly journals Role of phospholipid in the intermediate steps of the sodium-plus-potassium ion-dependent adenosine triphosphatase reaction

1975 ◽  
Vol 146 (3) ◽  
pp. 729-738 ◽  
Author(s):  
K P Wheeler
Keyword(s):  
Phosphatase Activity ◽  
Adenosine Triphosphatase ◽  
Atp Hydrolysis ◽  
Reaction Sequence ◽  
Single Extraction ◽  
Background Value ◽  
Dependent Atpase ◽  
Membrane Atpase ◽  
Normal Enzyme

The phosphorylation and dephosphorylation steps of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) reaction have been compared in ‘normal’, lipid-depleted and ‘restored’ membrane ATPase preparations. Partial lipid depletion was achieved by a single extraction with Lubrol W, and ‘restoration’ by adding pure phosphatidylserine. γ-32-P-labelled ATP was used for phosphorylation. The main findings were as follows. (1) Partial lipid depletion decreased but did not prevent Na-+-dependent phosphorylation, although it virtually abolished both Na-+-dependent and (Na-++K-+)-dependent ATPase activities. (2) ‘Restoration’ with phosphatidylserine produced an increment in phosphorylation that was the same in the presence and absence of added Na-+. (3) K-+ decreased the extent of Na-+-dependent phosphorylation of the depleted enzyme without producing a corresponding release of Pi. (4) K-+ rapidly decreased the extent of phosphorylation of the ‘restored’ enzyme to near-background value, with a concomitant release of Pi. (5) Na-+-dependent ATP hydrolysis was not restored. (6) The turnover of the ‘restored’ enzyme seemed to be higher than that of the ‘normal’ enzyme. The reaction sequence is discussed in relation to these results and the fact that the depleted enzyme retained about 50% of K-+-dependent phosphatase activity.

scholarly journals Subcellular organization of neurophysins, oxytocin, [8−lysine]-vasopressin and adenosine triphosphatase in porcine posterior pituitary lobes

1973 ◽  
Vol 132 (3) ◽  
pp. 361-371 ◽  
Author(s):  
J. C. Pickup ◽  
C. I. Johnston ◽  
S. Nakamura ◽  
L. O. Uttenthal ◽  
D. B. Hope
Keyword(s):  
Density Gradient ◽  
Adenosine Triphosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Posterior Pituitary ◽  
Neurosecretory Granules ◽  
Sucrose Density Gradient Centrifugation ◽  
Lysine Vasopressin ◽  
Neurophysin Ii

Posterior pituitary lobes from young pigs were fractionated by differential and sucrose-density-gradient centrifugation. The distributions of oxytocin and [8-lysine]-vasopressin were measured by bioassay and the distributions of neurophysin-I and -II by radioimmunoassays specific for each of these two proteins. Most of the hormone and neurophysin applied to the density gradient was localized in particles with the density expected of neurosecretory granules. However, the neurosecretory granules were separated into two bands (D and E). A close statistical correlation between the distributions of [8-lysine]-vasopressin and neurophysin-I, and of oxytocin and neurophysin-II on the gradients, suggested that in vivo porcine neurophysin-I binds [8-lysine]-vasopressin within one population of granules and porcine neurophysin-II binds oxytocin within another type of granule. However, there was no significant separation of oxytocin and vasopressin in fractions D and E. The molar ratios of hormones and neurophysins indicated that there was insufficient of either neurophysin to bind the [8-lysine]-vasopressin in the granule fractions or in the whole gland. Polyacrylamide-gel electrophoresis showed that only bands corresponding in mobility to porcine neurophysins-I, -II and -III were present in large amounts in the whole gland and in the granule fractions. The component with the mobility of neurophysin-III was, however, relatively enriched in whole young glands and granule fractions compared with adult gland extracts. It is suggested that the vasopressin that cannot be assigned to neurophysin-I may occur in (a) vesicles containing vasopressin but no neurophysin, (b) vesicles containing vasopressin and a protein that cannot be quantified by the radioimmunoassays used, such as porcine neurophysin-III, or (c) normal vasopressin–neurophysin granules which have accumulated extra vasopressin. Band E of the gradient was rich in adenosine triphosphatase activity, whereas band D possessed very little of this enzyme.

scholarly journals Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

1979 ◽  
Vol 182 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Andrea Quaroni ◽  
Katharina Kirsch ◽  
Milton M. Weiser
Keyword(s):  
Golgi Complex ◽  
Membrane Fraction ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Membrane Glycoproteins ◽  
Intestinal Villus ◽  
Microvillus Membrane ◽  
Small Intestinal ◽  
Small Intestinal Villus

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-3H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.

scholarly journals Inhibition of RhoA Translocation and Calcium Sensitization by In Vivo ADP-Ribosylation with the Chimeric Toxin DC3B

1997 ◽  
Vol 8 (12) ◽  
pp. 2437-2447 ◽  
Author(s):  
Hideyoshi Fujihara ◽  
Lori A. Walker ◽  
Ming Cui Gong ◽  
Emmanuel Lemichez ◽  
Patrice Boquet ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Guanosine Triphosphate ◽  
Calcium Sensitization ◽  
Adp Ribosylation ◽  
Kinase C ◽  
Rabbit Portal Vein ◽  
Intact Rabbit ◽  
Sensitizing Effect ◽  
Tonic Phase

Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10−6 M, 48 h; Aullo et al., 1993 ; Boquet et al. 1995 ) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)γS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPγS-induced translocation of cytosolic RhoA ( Gonget al., 1997a ) to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPγS, which prevents immunoprecipitation of non-ADP–ribosylated RhoA. Dissociation of cytosolic RhoA–rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop ( Weiet al., 1997 ) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.

THE EFFECTS OF AGE AND OESTRONE TREATMENT ON DNA POLYMERASE ACTIVITY IN ANTERIOR PITUITARY GLANDS OF MALE RATS

1973 ◽  
Vol 59 (1) ◽  
pp. 107-119 ◽  
Author(s):  
ANDREA MASTRO ◽  
W. C. HYMER
Keyword(s):  
Dna Polymerase ◽  
Anterior Pituitary ◽  
Reaction Medium ◽  
Polymerase Activity ◽  
Male Rats ◽  
Synthetic Activity ◽  
Cytoplasmic Fraction ◽  
Isolated Nuclei ◽  
Pituitary Glands

SUMMARY DNA polymerase activity was found in the cytoplasmic fraction and in isolated nuclei from anterior pituitary glands of male rats. The enzyme activity was assayed by measuring the incorporation of [3H]dTTP into DNA in a medium containing Tris-HCl buffer (pH 8·5), the four deoxyribonucleoside triphosphates, Mg2 +, ATP and activated calf thymus DNA. The DNA polymerase activity decreased with age in glands from animals aged 25 days to over a year but increased after oestrone treatment in vivo. These changes in activity, more pronounced in the cytoplasmic fraction than in the isolated nuclei, were similar to changes in DNA synthesis measured in anterior pituitary glands under the same physiological conditions. Isolated nuclei also retained endogenous DNA synthetic activity in the absence of added template. Addition of a cytoplasmic fraction to the reaction medium stimulated activity by as much as 1·9-fold but the degree of stimulation was the same whether the cytoplasm was from young, old or oestrone-treated animals.

scholarly journals Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation.

1987 ◽  
Vol 105 (3) ◽  
pp. 1241-1251 ◽  
Author(s):  
J R Bartles ◽  
H M Feracci ◽  
B Stieger ◽  
A L Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Subcellular Fractionation ◽  
Aminopeptidase N ◽  
Rat Hepatocyte ◽  
Dipeptidylpeptidase Iv ◽  
Plasma Membrane Fraction ◽  
Apical Domain ◽  
Basolateral Plasma Membrane

We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates. Approximate half-times for arrival are in the range of 90-120 min for aminopeptidase N and dipeptidylpeptidase IV whereas only 15-20% of the mature radiolabeled HA 4 associated with the hepatocyte plasma membrane fraction has become apical even after 150 min of chase. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain, followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates.

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