Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements

The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity. We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis. Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity). On the other hand, the aspartic acid mutant (ED144) retained in vivo activity. Substrate binding and catalytic studies were done with purified ED144 enzyme. The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage. Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive. The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex.     

Specificity and pH dependence for acylproline cleavage by prolidase

Catalytic pH dependence for the hydrolytic activity of the enzyme prolidase with a series of dipeptide substrates is found to be generally bell-shaped (kcat/Km) or simple sigmoidal (kcat). An enzymic residue with a pKa value of 6.6 is found to be critically involved in the catalytic mechanism, as is the substrate amino group. Significant catalysis at a pH of 6.6 is also observed for prolidase with (alkylthio)acetylprolines and with haloacetylprolines. A reverse-protonation state mechanism for substrate binding and activation is postulated, involving a chelative interaction of the aminoacylamide portion of substrate with a strongly Lewis-acidic active site metal ion.     

Does the neurotropic action of adrenocorticotrophic hormone involve a lipolytic step?

The stimulatory effect of adrenocorticotrophic hormone (ACTH)-related synthetic peptides on the hydrolysis of emulsified trioleoylglycerol by a rat brain lipase was studied. The ACTH effect was related to the net positive charge associated with the basic amino acid residues at position 15-18 in the ACTH sequence, as well as to the presence of the NH2-terminal amino acid residues at position 1-2. The ACTH effect on lipolysis was markedly reduced when lipids were partially removed from the enzyme preparation by extraction with chloroform/acetone. Full restoration of the stimulatory effect was obtained upon addition of phosphatidylcholine (2 mg/ml) to the lipolytic medium. Striking similarities between the structure-activity pattern for the stimulatory effect of ACTH on brain lipase and that described for the receptor-mediated actions of ACTH on adrenal and fat cells suggest that the ACTH effect might involve recognition of a binding site associated with the brain enzyme. Complete log concentration response curves obtained with four ACTH analogs may also be regarded as simulating hormone-receptor interaction. These findings are discussed in relation to the possibility that ACTH may have a neurohormonal role via lipase-catalyzed changes in the lipid matrix of membranes.     

Subunit interaction during catalysis. ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction

A new approach for assessing of catalytic cooperativity may occur between subunits has been applied to succinyl-CoA synthetase. This is based on the extent of oxygen exchange between medium [18O]Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis. Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium. With the Escherichia coli enzyme, which has an alpha 2 beta 2 structure, a pronounced increase in oxygen exchange per ATP cleaved occurs as ATP concentration is lowered. In contrast, when the CoA concentration is varied, the oxygen exchange per molecule of product formed remains constant. Also, with the pig heart enzyme, which is shown to retain its alpha beta structure during catalysis and thus has only one catalytic site, no modulation of oxygen exchange by ATP concentration is observed. These experimental findings show that the binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange. Measurement of the distribution of [18O]Pi species found as exchange occurs shows that only one catalytic sequence is involved in exchange at various ATP concentrations. These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at one catalytic site promotes catalytic site promotes catalytic events at an alternate catalytic site.     

Conformation and activity of chloroplast coupling factor exposed to low chemical potential of water in cells.

(1) Photophosphorylation, Ca2+-ATPase and Mg2+-ATPase activities of isolated chloroplasts were inhibited 55--65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (psi w) of --25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves. (2) These losses in activity were reflected in coupling factor (CF1) isolated from the leaves, since CF1 from leaves with low psi w had less Ca2+-ATPase activity than control CF1 and did not recouple phosphorylation in CF1-deficient chloroplasts. In contrast, CF1 from leaves having high psi w only partially recoupled phosphorylation by CF1-deficient chloroplasts from leaves havig low psi w. This indicated that low psi w affected chloroplast membranes as well as CF1 itself. (3) Coupling factor from leaves having low psi w had the same number of subunits, and the same electrophoretic mobility, and could be obtained with the same yields as CF1 from control leaves. However, direct measurements of fluorescence polarization, ultraviolet absorption, and circular dichroism showed that CF1 from leaves having low psi w differed from control CF1. The CF1 from leaves having low psi w also had decreased ability to bind fluorescent nucleotides (epsilon-ATP and epsilon-ADP). (4) Exposure of isolated CF1 to low psi w in vitro by preincubation in sucrose-containing media inhibited the Ca2+-ATPase activity of the protein in subsequent assays without sucrose. Inclusion of 5 or 10 mM Mg2+ in the preincubation medium markedly inhibited Ca2+-ATPase activity. (5) These results show that CF1 undergoes changes in cells which alter its phosphorylating ability. Since low cell psi w changed the spectroscopic properties but not other protein properties of CF1, the changes were most likely caused by altered confurn, photophosphorylation. The inhibition of ATPase activity in CF1 in vitro at low psi w and high ion concentration mimicked the change in activity seen in vivo.     

Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

During the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ～40,000) is observed. Enucleation of [³²P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone-induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [³²P] phosphoproteins.     

Effect of actin concentration on the intermediate oxygen exchange of myosin; relation to the refractory state and the mechanism of exchange.

The effect of actin concentration on the myosin catalyzed exchange of phosphate oxygens with water accompanying ATP hydrolysis has been investigated. The extent of exchange was found to extrapolate to zero at infinite actin concentration at 23 and 0 degrees C for myosin subfragments S1(A1) and S1(A2). This result is consistent with actin associating directly with the product of the hydrolysis step and is not readily consistent with refractory state schemes in which the entire flow goes via a dissociating pathway. The possibility of a refractory state in the form of a phosphorylated intermediate or a bound metaphosphate state with hydrolysis occurring in the transition to the refractory state merits consideration. A full analysis of the dependence of intermediate exchange on the rate constants of the acto-S1 scheme is given and the errors arising from other methods of analysis are discussed. The rate of oxygen exchange was measured as 10 s-1 (23 degrees C) a value comparable with but slightly lower than the rate of reversal of the ATP cleavage step.     

Structure and activity of chloroplasts of sunflower leaves having various water potentials

Summary Changes in membrane integrity, conformation and configuration, and in photosystem II (PS II) activity (measured as dichloroindophenol photoreduction) of sunflower (Helianthus annuus L.) chloroplasts were studied after leaf tissue had been desiccated to various water potentials ( _w ). Fixatives for electron microscopy were adjusted osmotically to within 1 bar of the _w of the tissue to prevent rehydration during fixation. PS II activity decreased to 50% of the control activity at a _w of-26 bar. At this _w , leaf viability was being lost but there was virtually no loss of integrity of the thylakoid lamellar system. Even at extreme _w (below-100 bar), thylakoids retained much structural detail but were less stained. At-26 bar, intrathylakoid spacing (configuration) and lamellar thickness (conformation) were decreased in vivo. Upon isolation of the plastids, the differences in configuration disappeared but the differences in conformation remained. The decreases in membrane conformation and PS II activity both, in vivo and in vitro suggest that alterations in conformation may cause decreases in chloroplast activity at _w as low as-26 bar. Since structural detail was maintained, however, previous observations of altered membrane integrity, which involved tissue fixed without osmotic support, may have been affected by tissue rehydration during fixation.Abbreviations DCIP sodium 2,6-dichloroindophenol - PS II photosystem II - _w leaf water potential     

The rapid labeling of adenosine triphosphate by 32P-labeled inorganic phosphate and the exchange of phosphate oxygens as related to conformational coupling in oxidative phosphorylation.

Evidence is presented that extends and amplifies the concept that in oxidative phosphorylation energy input serves to bring about release of ATP formed at a catalytic site by reversal of hydrolysis. The evidence with beef heart submitochondrial particles includes additional demonstration of uncoupler insensitive Pi leads to HOH exhchange, demonstration that this exchange is sensitive to the specific phosphorylation inhibitor, oligomycin, and demonstration that the small burst of uncoupler-insensitive ATP, rapidly labeled after addition of a tracer of 32Pi, behaves in a manner consistent with its participation as a membrane-bound intermediate in the Pi leads to HOH exchange. In addition, data are presented showing that addition of hexokinase plus glucose to submitochondrial particles in presence of ADP and Pi considerably lowers the Pi leads to HOH exchange but that further addition of cyanide or 2,4-dinitrophenol or both has little additional effect. Such data are compatible with no energy requirement for formation of bound ATP. However, with a large excess of hexokinase, the rate of the Pi leads to HOH exchange is further depressed. This could reflect some use of energy to promote formation of ATP at the catalytic site or to maintain the integrity of the phosphorylation system. Relationships of these findings to related information in the field are discussed.     

Serum hormone levels following partial hepatectomy in the rat.

Serum levels of insulin, glucagon, growth hormone (somatotrophin) and thyroxine (TT₄) were measured by radioimmunoassay following both sham operation and 70% partial hepatectomy in the rat to evaluate changes in hormone levels during liver regeneration. An eleven fold increase in glucagon was observed (from 112 ± 10 pg/ml to 1500 ± 200 pg/ml) 6 hours following partial hepatectomy but not sham operation. In contrast, insulin levels remained unchanged compared to sham controls for up to 72 hr while growth hormone fell to low levels, 6 to 48 hr after partial hepatectomy. Both total thyroxine and free thyroxine levels also fell 24–72 hours after hepatectomy. These studies suggest that growth hormone, thyroxine and insulin are not primary stimulants of hepatic regeneration although the data suggests that glucagon may modify this growth process.