scholarly journals Heterogeneous phosphorylation of erythrocyte spectrin β chain in intact cells

1993 ◽  
Vol 294 (3) ◽  
pp. 841-846 ◽  
Author(s):  
S Pedroni ◽  
M C Lecomte ◽  
H Gautero ◽  
D Dhermy
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Beta Chain ◽  
Intact Cells ◽  
Molecular Masses ◽  
Alpha Beta ◽  
Self Association

Human erythrocyte spectrin is an alpha beta heterodimer which forms tetramers by self-association. This association involves the N-terminal region of the alpha chain and the C-terminal region of the beta chain. The latter contains a cluster of four phosphorylation sites (one phosphothreonine and three phosphoserine residues). The role of this phosphorylation is as yet unknown. We show in this paper that the spectrin beta chain occurs in the cell in subpopulations differing in the degree of occupancy of their phosphorylation sites: 32P peptide maps obtained by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage revealed the presence of six components with apparent molecular masses of 17.5 kDa, differing in their isoelectric points; this is most simply interpreted as reflecting the presence of six exchangeable phosphorylation sites in the spectrin beta chain, rather than four as had been supposed. When the alpha beta dimers were partly dissociated by urea, the most highly phosphorylated fraction of the beta chain was found in the undissociated dimers. This high specific activity in the undissociated dimer reflected multiple phosphorylated sites, as revealed by NTCB cleavage. The dephosphorylation or the hyperphosphorylation of spectrin beta chains did not modify the equilibrium between dissociated and undissociated spectrin dimers in the presence of urea. However, the data revealed the existence of two spectrin dimer populations in respect to phosphate turnover and spectrin dimer dissociation.

Role of vacuoles and vesicles in extracellular enzyme secretion from yeast

1973 ◽  
Vol 19 (1) ◽  
pp. 113-117 ◽  
Author(s):  
R. A. Holley ◽  
D. K. Kidby
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Extracellular Enzyme ◽  
Invertase Activity ◽  
Preliminary Evidence ◽  
Acid Protease ◽  
Intracellular Enzyme ◽  
Per Se ◽  
Equilibrium Centrifugation

Preliminary evidence has been obtained which suggests that the intracellular invertase of Saccharomyces cerevisiae may not be localized in the vacuole per se. Alkaline phosphatase, an intracellular enzyme, and acid protease, a typically lysosomal enzyme, both showed high specific activity in the vacuole fraction prepared by equilibrium centrifugation of lysed sphaeroplasts in Ficoll gradients. Invertase activity has been found to be associated with vacuoles only when glucose-repressed cells are derepressed. Cells derepressed for invertase biosynthesis contained a population of vesicles which were virtually absent from the repressed cells. Evidence is presented which strongly suggests that these vesicles rather than the vacuoles are the vehicle by which invertase is secreted from the cell.

scholarly journals Both Subunits of ATP-Citrate Lyase from Chlorobium tepidum Contribute to Catalytic Activity

2006 ◽  
Vol 188 (18) ◽  
pp. 6544-6552 ◽  
Author(s):  
Wonduck Kim ◽  
F. Robert Tabita
Keyword(s):  
Catalytic Mechanism ◽  
Specific Activity ◽  
High Specific Activity ◽  
Small Subunit ◽  
High Yield ◽  
Chlorobium Tepidum ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Green Sulfur

ABSTRACT ATP-citrate lyase (ACL) is an essential enzyme of the reductive tricarboxylic acid (RTCA) pathway of CO2 assimilation. The RTCA pathway occurs in several groups of autotrophic prokaryotes, including the green sulfur bacteria. ACL catalyzes the coenzyme A (CoA)-dependent and MgATP-dependent cleavage of citrate into oxaloacetate and acetyl-CoA, representing a key step in the RTCA pathway. To characterize this enzyme from the green sulfur bacterium Chlorobium tepidum and determine the role of its two distinct polypeptide chains, recombinant holo-ACL as well as its two individual subunit polypeptides were synthesized in Escherichia coli. The recombinant holoenzyme, prepared from coexpressed large and small ACL genes, and the individual large and small subunit polypeptides, prepared from singly expressed genes, were all purified to homogeneity to high yield. Purified recombinant holo-ACL was isolated at high specific activity, and its k cat was comparable to that of previously prepared native C. tepidum ACL. Moreover, the purified recombinant large and small subunit polypeptides were able to reconstitute the holo-ACL in vitro, with activity levels approaching that of recombinant holo-ACL prepared from coexpressed genes. Stoichiometric amounts of each subunit protein were required to maximize the activity and form the most stable structure of reconstituted holo-ACL. These results suggested that this reconstitution system could be used to discern the catalytic role of specific amino acid residues on each subunit. Reconstitution and mutagenesis studies together indicated that residues of each subunit contributed to different aspects of the catalytic mechanism, suggesting that both subunit proteins contribute to the active site of C. tepidum ACL.

scholarly journals Localization of sites of enhanced expression of endothelin-1 in the kidney of DOCA-salt hypertensive rats.

1996 ◽  
Vol 7 (8) ◽  
pp. 1158-1164 ◽  
Author(s):  
L Y Deng ◽  
R Day ◽  
E L Schiffrin
Keyword(s):  
Mesangial Cells ◽  
Specific Activity ◽  
High Specific Activity ◽  
Hypertensive Rats ◽  
Endothelin 1 ◽  
Renal Vessels ◽  
Enhanced Expression ◽  
Potent Vasoconstrictor

Although the role of endothelin-1, a potent vasoconstrictor peptide, in hypertension remains unclear, there is evidence of its involvement in deoxycorticosterone acetate (DOCA)-salt hypertensive rats, in which enhanced vascular production of endothelin-1 has been documented. The study presented here examined endothelin-1 gene expression in the kidney in DOCA-salt hypertensive rats by in situ hybridization histochemistry. A high specific activity 35S-labeled complementary RNA probe was used. Significant increases in abundance of endothelin-1 mRNA transcripts were found in the endothelium of renal vessels, and in capillary endothelial and mesangial cells of glomeruli of the remaining kidney of DOCA-salt hypertensive rats, in comparison with unilaterally nephrectomized control rats. Enhanced expression of the endothelin-1 gene in the kidney of DOCA-salt hypertensive rats may participate in abnormalities of renal function in this model of hypertension, and thus contribute to the development and maintenance of elevated blood pressure.

scholarly journals Klebsiella pneumoniae nitrogenase MoFe protein: chymotryptic proteolysis affects function by limited cleavage of the β-chain and provides high-specific-activity MoFe protein

1993 ◽  
Vol 291 (1) ◽  
pp. 309-314
Author(s):  
K Fisher ◽  
D J Lower ◽  
R N Pau
Keyword(s):  
Klebsiella Pneumoniae ◽  
Specific Activity ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Visible Spectrum ◽  
Prolonged Treatment ◽  
Beta Chain ◽  
Wild Type ◽  
Alpha Chain ◽  
Mofe Protein

Proteinase treatment with chymotrypsin has been used to probe the structure of native Klebsiella pneumoniae nitrogenase MoFe protein (Kp1). Reaction with chymotrypsin did not bleach Kp1, suggesting that it did not destroy the metal centres, and the Mo and Fe contents of Kp1 were unchanged. High ratios of chymotrypsin to Kp1 (1:1 by mass) cleaved the beta-chain of Kp1 to give 44 and 14 kDa polypeptides, which N-terminal amino acid sequence analysis showed to be derived from cleavage at residue beta-Phe124. A mutant MoFe protein, Kp1Met-124, in which beta-Phe124 is replaced by methionine, was not cleaved by chymotrypsin. Under non-denaturing conditions, the ‘nicked’ beta-chain of the wild-type protein remained associated with the alpha-chain. The alpha-chain was not cleaved by the proteinase treatment. Fission of the wild-type beta-chain was accompanied by loss of enzyme activity, loss of intensity of the g = 3.7 e.p.r. signal derived from dithionite-reduced FeMoco and by changes in the visible spectrum. The e.p.r. spectra of potassium ferricyanide-oxidized native and digested Kp1 show differences in the signals between g = 1.6 and 2.0. After prolonged treatment, the final specific activity of Kp1 was about 25 +/- 5% of the initial activity. This corresponded to 25 +/- 5% of the beta-chain which was resistant to proteolytic action. Brief treatment of Kp1 with a lower concentration of chymotrypsin (chymotrypsin/Kp1 ratio = 1:10 by mass, for 10 min) preferentially cleaved high-molecular-mass polypeptides that routinely contaminate preparations of Kp1 prepared by standard procedures. Treatment with chymotrypsin followed by gel filtration to remove the proteinase and cleaved protein fragments can therefore be used to increase significantly the specific activity of Kp1 preparations and remove contaminating activities, such as the ATPase activity of myokinase.

Role of pancreatic L-asparagine synthetase in homeostasis of L-asparagine.

1979 ◽  
Vol 236 (6) ◽  
pp. E746 ◽  
Author(s):  
H A Milman ◽  
D A Cooney ◽  
D M Young
Keyword(s):  
Amino Acid ◽  
Exocrine Pancreas ◽  
Specific Activity ◽  
Pancreatic Enzyme ◽  
High Specific Activity ◽  
Asparagine Synthetase ◽  
Mouse Pancreas ◽  
Normal Limits ◽  
Intravenous Treatment

L-Asparagine synthetase from mouse pancreas was found to be associated principally with the exocrine pancreas and to be dependent on the age of the animal, but not on gender, diet, or the presence of tumor under the conditions examined. The function of the pancreatic enzyme appears to be to supply L-asparagine for the synthesis of pancreatic proteins. This function is suggested by the high specific activity of L-asparagine in pancreatic proteins after intravenous treatment of BDF1 mice with L-[U-14C]asparatate. The pancreas is also able to function as a storage depot for L-asparagine under conditions in which the concentration of the amino acid in the blood is in excess. Unlike the liver, the pancreas is unable to add L-asparagine to the circulation when the concentration of the amide is below normal limits.

Are Spinach Chloroplasts Involved in Flavonoid O-Methylation?

1985 ◽  
Vol 40 (5-6) ◽  
pp. 331-335 ◽  
Author(s):  
Kim Thresh ◽  
Ragai K. Ibrahim
Keyword(s):  
Specific Activity ◽  
Specific Binding ◽  
Plant Secondary Metabolites ◽  
High Specific Activity ◽  
Spinach Chloroplasts ◽  
Comt Activity ◽  
Activity Ratios ◽  
Methylation Patterns ◽  
Spinach Leaves

Abstract Intact spinach chloroplasts, which were isolated on a cushion of Percoll and purified on discon­tinuous sucrose gradients, were found to adsorb appreciable amounts of high specific activity, cytosolic O-methyltransferases. The latter activity was readily eliminated after washing the chloroplast pellet with 0.1 ᴍ phosphate in sucrose gradient buffer and finally with 0 .1% nonidet. Furthermore, the OMT activity of spinach leaves was resolved by chromatography on DEAE-Sepharose CL-6B into caffeic acid (COMT) and flavonoid (FOMT) O-methyltransferase ac­tivities, and purified to 30-and 50-fold, respectively. The similarities between the FOMT/COMT activity ratios and the methylation patterns of intact chloroplasts and their supernatants, as well as those of purified leaf preparations, suggest non-specific binding of cytosolic OMTs to chloroplast envelopes. It is concluded, therefore, that spinach chloroplasts are not involved in the methylation of phenylpropanoid or flavonoid compounds. These results call attention for the re-evaluation of the role of this organelle in the biosynthesis or accumulation of plant secondary metabolites.

scholarly journals The Catastrophe-promoting Activity of Ectopic Op18/Stathmin Is Required for Disruption of Mitotic Spindles But Not Interphase Microtubules

2001 ◽  
Vol 12 (1) ◽  
pp. 73-83 ◽  
Author(s):  
Per Holmfeldt ◽  
Niklas Larsson ◽  
Bo Segerman ◽  
Bonnie Howell ◽  
Justin Morabito ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Polymerization Rate ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Mitotic Spindles ◽  
Intact Cells ◽  
Spindle Formation ◽  
Tubulin Binding

Oncoprotein18/stathmin (Op18) is a microtubule (MT) destabilizing protein that is inactivated during mitosis by phosphorylation at four Ser-residues. Op18 has at least two functions; the N-terminal region is required for catastrophe-promotion (i.e., transition from elongation to shortening), while the C-terminal region is required to inhibit MT-polymerization rate in vitro. We show here that a “pseudophosphorylation” derivative of Op18 (i.e., four Ser- to Glu-substitutions at phosphorylation sites) exhibits a selective loss of catastrophe-promoting activity. This is contrasted to authentic phosphorylation, which efficiently attenuates all activities except tubulin binding. In intact cells, overexpression of pseudophosphorylated Op18, which is not phosphorylated by endogenous kinases, is shown to destabilize interphase MTs but to leave spindle formation untouched. To test if the mitotic spindle is sensitive only to the catastrophe-promoting activity of Op18 and resistant to C-terminally associated activities, N- and C-terminal truncations with defined activity-profiles were employed. The cell-cycle phenotypes of nonphosphorylatable mutants (i.e., four Ser- to Ala-substitutions) of these truncation derivatives demonstrated that catastrophe promotion is required for interference with the mitotic spindle, while the C-terminally associated activities are sufficient to destabilize interphase MTs. These results demonstrate that specific Op18 derivatives with defined activity-profiles can be used as probes to distinguish interphase and mitotic MTs.

Role of anions and carbonic anhydrase in epithelia

1982 ◽  
Vol 299 (1097) ◽  
pp. 369-381 ◽  
Keyword(s):  
Carbonic Anhydrase ◽  
Epithelial Transport ◽  
Carbonic Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
Salt Transport ◽  
Biliary Duct ◽  
Toad Urinary Bladder ◽  
Hydration And Dehydration

The existence of carbonic anhydrase (carbonate dehydratase, EC 4. 2.1.1) in blood was suspected and sought because the rates of spontaneous hydration and dehydration of CO 2 and carbonic acid were slow compared with the rates of exchange of CO 2 with blood. The existence of the enzyme in absorbing and secreting epithelial tissues has, in contrast, often been sought because its presence was required for the operations of theoretical models for the movements of H + ions or HCO - 3 into or out of epithelial cells. In addition to the HCl-secreting gastric mucosal epithelium, the enzyme was subsequently found in the rumen, in the kidney, especially those of species that produce acid urine, in salivary gland, the liver and biliary duct system, the mucosa of the small intestine, caecum and colon, the choroid plexuses and ciliary body of mammals, in toad urinary bladder and in the Cl - secreting cells of fish gill. The presence of carbonic anhydrase in exocrine pancreas does not seem to be well established. The enzyme, of molecular mass about 30 kDa and containing one zinc atom, exists in three related forms: one of high specific activity and two of low specific activity, one of which is found in red skeletal muscle. Although most, but not all, types of erythrocyte contain both varieties, epithelia usually contain only the highactivity enzyme; however, ox rumen contains large quantities of the low-activity variety as do guinea-pig caecal and colonic mucosae. Salt transport in the intestinal tract is associated with movements of HCO - 3 and of H + ions, yet although carbon dioxide stimulates solute and fluid transport in the gall bladder and jejunum, and inhibitors of carbonic anhydrase reduce fluid and ion transport across many epithelia, the role of the enzyme in epithelial transport is not clearly understood. Knowledge of the rates of hydration and dehydration of CO 2 /HCO - 3 in the fraction of the tissue water responsible for the H + -HCO - 3 movements in many secretory epithelia is currently lacking.

scholarly journals Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches

1980 ◽  
Vol 186 (3) ◽  
pp. 713-723 ◽  
Author(s):  
F Mollinedo ◽  
V Larraga ◽  
F J Coll ◽  
E Muñoz
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Relative Proportion ◽  
Inhibitory Effect ◽  
Beta Subunit ◽  
Gamma Subunit ◽  
Micrococcus Lysodeikticus ◽  
Alpha Beta

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.

scholarly journals Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity

1990 ◽  
Vol 270 (1) ◽  
pp. 17-26 ◽  
Author(s):  
P I Bauer ◽  
K G Buki ◽  
A Hakam ◽  
E Kun
Keyword(s):  
Protein Concentration ◽  
Specific Activity ◽  
Enzymic Activity ◽  
Cross Linking ◽  
Enzyme Protein ◽  
Protein Fluorescence ◽  
Molecular Masses ◽  
Self Association ◽  
Adp Ribosyltransferase ◽  
Macromolecular Association

The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3′-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.

Sign in / Sign up

Export Citation Format

Share Document