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At least 19 recordsLinked to original sources
PubMed1977

Effects of cyclic nucleotides on the conformational states of the alpha core of the cyclic AMP receptor protein.

The alpha core gragment produced by limited proteolysis contains the cyclic AMP binding domain and the two buried sulfhydryl groups of the cyclic AMP receptor protein. The buried sulfhydryl groups of the alpha core react with 5,5'-dithio-bis(2-nitrobenzoic acid) after denaturation by 3 M urea or digestion with subtilisin. The rate of sulfhydryl modification in the presence of 3 M urea or subtilisin is markedly decreased in the presence of cyclic nucleotides which are proposed to tighten the conformation of the alpha core. Incubation of the alpha core in 3 M urea or dithionitrobenzoic acid does not affect cyclic AMP binding while dithionitrobenzoic acid plus 3 M urea inhibits cyclic AMP binding suggesting a role for the buried sulfhydryls in cyclic AMP binding or their proximity to the cyclic AMP binding domain of the alpha core. The data are consistent with a ligand-induced conformational change in the alpha region of the native cyclic AMP receptor protein that is required for DNA binding.

Binding Sites↗
PubMed1977

Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation.

The modification of Escherichia coli citrate synthase (citrate oxaloacetatelyase(pro-3S-CH2.COO- leads to acetyl-CoA, EC 4.1.3.7) with 5,5'-dithiobis-(2-nitrobenzoic acid) has been investigated. (1) In low ionic strength (20 mM Tris.HCl, pH 8.0): (A) Eight thiol groups per tetramer of the native enzyme reacted with Nbs2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete densensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols is 2.5.10(4) M-1.min-1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. (2) In high ionic strength (20 mM Tris.HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. (3) From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response of NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possessing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.

Chemical Phenomena↗
PubMed1977

Cleavage of the S-S bond in 5,5'-dithiobis-(2-nitrobenzoic acid) in the presence of a cationic detergent: an approach to the cleavage of the S-S bond in bovine plasma albumin.

The hydrolysis of 5,5'-dithiobis-(2-nitrobenzoic acid) was studied at pH 9.20 and 25 degrees C in presence of tetradecyltrimethylammonium bromide or micellar tetradecyltrimethylammonium bromide. The reaction was pseudo-unimolecular reaction with regard to the concentration of dithionitrobenzoic acid. The rate constant k1 depended on the tetradecyltrimethylammonium bromide concentration and on the ionic strength of the buffer solution. At a constant ionic strength, the value of k1 increased with the increase in the tetradecyltrimethylammonium bromide concentration, attained maximum at a certain concentration above the critical micelle concentration, and then decreased. The value of k1 was larger when the ionic strength was lower. The rate of hydrolysis at the ionic strength 0.1 was the same as that without tetradecyltrimethylammonium bromide. On the contrary, sodium dodecyl sulfate or micellar sodium dodecyl sulfate had no effect on the rate of hydrolysis. These results lead to the conclusion that the S-S bond is cleaved easier, when it is surrounded by the cationic detergent. The SH/S-S exchange reaction of bovine plasma albumin in the presence of cationic detergent was slower when the ionic strength was higher. The fact could be explained by assuming that the exposed S-S bond is surrounded by the cationic detergent. Further, it was speculated that some S-S bonds in albumin are surrounded by the positively charged basic amino acid residues.

Detergents↗
PubMedDate not supplied

Quantification and localisation of SH-groups in human blood serum proteins.

The thiol groups of human blood serum proteins were determined after 24 hours interaction with dithionitrobenzoic-acid (DTNB) to an average of 538 +/- 60 mumol/l serum. After treatment of the serum with [35S]DTNB, autoradiograms of the protein elpherograms revealed two main peaks: The first with 63% of total activity, in the albumin region, corresponding to 0.60 SH/mol, the second with 23% of total activity, in the gamma-globulin range, corresponding to 2.2 SH/mol. After 30 minutes incubation with DTNB, or with p-chloromercuribenzoate (CMB), in freshly prepared pools of IgG only 0.2 SH/mol were found which is the expected value already known from the literature. Autoradiograms taken from serum protein elpherograms after interaction with [14C]CMB only show the main SH-peak in the albumin range. Thus it is concluded that the SH-peak in the gamma-globulin region after 24 hours incubation with [35S]DTNB is due to one highly labile S-S-bond which easily undergoes a disulfide exchange with DTNB.

Blood Proteins↗
PubMed1976

Discoidal bilayer structure of nascent high density lipoproteins from perfused rat liver.

Rat livers were perfused for 6 h without added plasma proteins using washed erythrocytes and buffer in a recirculating system. An inhibitor to the enzyme lecithin-cholesterol acyltransferase (5,5'-dithionitrobenzoic acid) was added in some experiments to prevent modification of substrate-lipids contained in secreted lipoproteins. The inhibitor did not detectably alter hepatic ultrastructure or gas exchange, but it inhibited the secreted lecithin-cholesterol acyltransferase by more than 85%. Very low density lipoproteins in perfusate were unaltered but the high density lipoproteins obtained from livers perfused with the inhibitor appeared disk-shaped in negative stain by electron microscopy with a mean edge thickness of 46 +/- 5 A and a mean diameter of 190 +/- 25 A. The high density lipoproteins were composed predominantly of polar lipids and protein with only small amounts of cholesteryl esters and triglycerides. The major apoprotein of these discoidal fractions had the same electrophoretic mobility as the arginine-rich apoprotein, whereas plasma high density lipoproteins contained mainly the A-I approtein. In all these respects the discoidal perfusate high density lipoproteins closely resemble those found in human plasma which is deficient in lecithin-cholesterol acyltransferase. Perfusate high density lipoproteins obtained in the absence of the enzyme inhibitor more closely resembled plasma high density lipoproteins in chemical composition (content of cholesteryl esters and apoproteins) and in electron microscopic appearance. Purified lecithin-cholesterol acyltransferase synthesized cholesteryl esters at a substantially faster rate from substrate lipids of perfusate high density lipoproteins than those from plasma. The discoidal high density lipoproteins were the best substrate for this reaction. Thin sections of plasma high density lipoproteins indicated a spherical particle whereas discoidal high density lipoproteins stained with the characteristic trilaminar image of membranes. These observations suggest that the liver secretes disk-shaped lipid bilayer particles which represent both the nascent form of high density lipoproteins and preferred substrate for lecithin-cholesterol acyltransferase.

Animals↗
PubMed1979

Distribution of sulfhydryl groups in intestinal brush border membranes. Localization of side-chains essential for glucose transport and phlorizin binding.

1. Brush border membrane vesicles from rabbit small intestine were found to contain 46 nmol SH groups/mg protein, 52% of which could react with 4,4'-dithiodipyridine, a membrane permeating probe. Only 18% of the total SH-groups reacted with the impermeant probe 5,5'-dithiobis(2-nitrobenzoic acid), indicating that only this fraction is externally located. 2. Brush border membrane vesicles could be disrupted by a gentle treatment with deoxycholate, releasing most of their electron-dense core material. In deoxycholate-treated vesicles most of the SH groups that reacted with 4,4'-dithiodipyridine react with 5,5'-dibiobis(2-nitrobenzoic acid), suggesting that both membrane surfaces became exposed to the extravesicular medium. 3. In intact vesicles (1.2 mg protein/ml), the binding of phlorizin (a competitive inhibitor of the monosaccharide transport system) was 50% inhibited by 67 microM of the penetrating organomercurial p-chloromercuribenzoate, but was about ten times less sensitive to the poorly permeating p-chloromercuriphenylsulfonate. In contrast, binding of phlorizin to leaky (deoxycholate-treated) membranes was equally susceptible to either reagent. 4. Mercurial inhibition of phlorizin binding could be reversed by dithioerythritol in both sealed and leaky membranes, whereas the less permeant thiol L-glutathione (reduced form) could only revert the inhibition in leaky membranes.

Animals↗
PubMed1978

Rat intestine secretes discoid high density lipoprotein.

High density lipoprotein was isolated from pooled rat serum and mesenteric lymph of lymph fistula rats. In most experiments, 5,5'-dithionitrobenzoic acid, an inhibitor of the enzyme lecithin:cholesterol acyltransferase, was added during the collection of lymph to prevent modification of the lipid composition of newly secreted lipoproteins. Negative staining electron microscopy of lymph high density lipoprotein revealed discoidal particles (190+/-3 x 55+/-1 A) which tended to form rouleaux, smaller spherical particles were also present. Serum high density lipoprotein contained only spherical particles (diameter 93+/-4 A). Lipid analysis showed that lymph high density lipoprotein was enriched in phospholipid and deficient in cholesterol esters when compared to serum high density lipoprotein. The phospholipid to cholesterol esters ratio was greatest in basal lymph high density lipoprotein when compared to fatty lymph and serum high density lipoprotein. From analysis of the lipid compositional data and direct particle measurement by electron microscopy, it could be determined that congruent with50% of basal lymph high density lipoprotein and 30% of fatty lymph high density lipoprotein was discoid. Basal lymph high density lipoprotein was enriched in apoA-I and deficient in the arginine-rich peptide, and the apoprotein composition of fatty lymph high density lipoprotein more closely resembled serum. These observations demonstrate that intestinal lymph contains two types of high density lipoprotein particles, a discoid nascent particle deficient in cholesterol ester and rich in apoA-I, and spherical high density lipoprotein derived from plasma. A significant amount of lymph high density lipoprotein appears to be secreted by the intestine.

Animals↗
PubMedDate not supplied

[Interaction of platinum and palladium complexes with thiol groups of Ca2+-dependent ATPase from sarcoplasmic reticulum].

In native preparations of sarcoplasmic reticulum 10-12 thiol groups (in g-eqv per 10(5) g of protein) were estimated by the Benesh method (titration with AgNO3) and 2 thiol groups--by Ellman (titration with dithionitrobenzoic acid). After denaturation of the sarcoplasmic reticulum preparations with 8 M urea 10-12 thiol groups were also determined by the Ellman method. When the preparations were treated with platinum tetrachloride or with palladium diaminodichloride, only 3 thiol groups were estimated by the Benesh and no one - by the Ellman method. Platinum and palladium complexes inhibited also the Ca2+-dependent activity blocking the transport of Ca2+ in sarcoplasmic reticulum. The inhibition was partially removed by glutathione.

Adenosine Triphosphatases↗
PubMed1977

Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.

Animals↗
PubMedDate not supplied

[Possibilities for Ellman reaction application in the presence of detergents].

Triton X-100, triton X-305, twin-80 and sodium deoxycholate in definite concentrations lower the colour intensity of the solution which contains the product of the Ellman reaction. In the case of triton X-100 the colour disappearance is due to resynthesis of the Ellman reagent from 5-thio-2-nitrobenzoate with the presence of detergent in the concentration which is higher than the critical concentration of micelle-formation.

Acetylcholinesterase↗
PubMed1977

Monkey glutathione S-aryltransferases. II. Properties of the major enzyme purified from the liver.

1. The molecular and enzymatic properties of the major component (Fraction IV) of glutathione S-aryltransferases [EC 2.5.1.13] purified from the liver of monkey (mainly rhesus monkey) have been investigated. The enzyme had a molecular weight of about 48,000 and was composed of two subunits of apparently identical molecular weight (ca. 24,000) bound to each other non-covalently. Each subunit contained one SH group. The amino acid composition showed characteristic high contents of leucine and glutamic acid residues. No amino-terminal residue was detected by the dansyl method. 2. The enzyme showed a rather broad optimum pH range from 7.5 to 9 with 1,2-dichloro-4-nitrobenzene as a substrate. It was moderately stable below 40 degrees C at pH 7.5. However, it showed an anomalous instability at pH around 4.2. It was reversibly denatured at least partially by urea or guanidine hydrochloride and irreversibly denatured by sodium dodecyl-sulfate. It was significantly inhibited by Zn2+, Cd2+, and Hg2+, and also by benzene hexachloride. It was extensively inactivated by reaction with phenylglyoxal or 2,4,6-trinitrobenzene sulfonate, whereas several SH reagents were without marked effect on the activity under the reaction conditions employed.

Amino Acids↗
PubMed1978

Homoserine dehydrogenase of Rhodospirillum rubrum. Physical and chemical characterization.

A detailed physicochemical characterization of purified homoserine dehydrogenase of Rhodospirillum rubrum is presented. The enzyme has a molecular weight of 110000 and consists of two subunits of identical molecular weight of 55000. Depending on the ionic strength and protein concentration it is possible for the native enzyme to dimerize to produce an enzymatically active species of molecular weight 220000. Titrations of the native and detergent-treated enzyme with a variety of sulfhydryl reagents show 2 mol free--SH groups per 110000 g, one of which is buried in the protein interior. L-Threonine and/or high concentrations of salt can expose the buried--SH group, and this--SH group is essential for the catalytic activity of the enzyme. Two independent lines of evidence show that extensive polymerization of the enzyme caused by L-threonine and/or high concentrations of salt does not involve the formation of intermolecular disulfide bonds.

Alcohol Oxidoreductases↗
PubMed1978

gamma-Glutamyl cyclotransferase from rat kidney. Sulfhydryl groups and isolation of a stable form of the enzyme.

gamma-Glutamyl cyclotransferase, highly purified from rat kidney, contains several readily accessible sulfhydryl groups whose modification appears to be associated with the appearance of multiple enzyme forms as determined by isoelectric focusing and ion exchange chromatography. The enzyme was obtained in a 1000-fold purified and apparently homogeneous form by a procedures involving treatment with dithiothreitol followed by chromatography on thiol-Sepharose. The enzyme was also isolated in a highly active, apparently homogeneous, and stable form after reduction and treatment with iodoacetamide. The amino acid compositions and other properties of the two forms of the enzyme were very similar. Studies on the activity of the enzyme toward a variety of gamma-glutamyl amino acids and di-gamma-glutamyl amino acids showed that the enzyme is much more active toward certain di-gamma-glutamyl amino acids than toward the corresponding gamma-glutamyl amino acids; thus, the preferred substrates have the general structure gamma-Glu-gamma-Glu-NH-R in which the nature of the R moiety has relatively little effect on activity.

Acyltransferases↗
PubMed1976

Biosynthesis of bacterial glycogen. Activator-induced oligomerization of a mutant Escherichia coli ADP-glucose synthase.

ADP-glucose synthase (EC 2.7.7.27) was purified to homogeneity from SG5-504, Aa mutant strain of Escherichia coli B. It had a molecular weight of approximately 2 X 10(5) and four identical subunits. In the presence of its allosteric activator, fructose-1-6-P2, the mutant enzyme formed oligomers with several times the tetramer molecular weight, as revealed by sedimentation equilibrium centrifugation, polyacrylamide gel electrophoresis, and gel filtration column chromatography. Enzyme purified from a wild type strain, AC70R1, did not exhibit the activator-induced oligomerization. Catalytically, the mutant enzyme had a 2-fold higher affinity for activator and a 2- to 3-fold lower affinity for the allosteric inhibitors 5'-adenylate and KH2PO4 than the wild type. The two enzymes appeared identical in subunit molecular weight, amino acid composition, COOH-terminal amino acid, and the first 27 residues of the NH2-terminal sequence. Subtle structural differences were revealed by chemical modification of specific residues. Compared to wild type, the mutant enzyme had 1 cysteine/subunit more accessible to modification by Ellman's reagent. SG5-504 enzyme was inactivated more slowly than AC70R1 enzyme by phenylglyoxal and by trinitrobenzenesulfonate. These results reflected an altered conformation of the SG5-504 enzyme molecule. The unique ability of it to oligomerize in the presence of activator may be the result of a single amino acid substitution.

Amino Acid Sequence↗
PubMed1976

Biosynthesis of bacterial glycogen. Characterization of the subunit structure of Escherichia coli B glucose-1-phosphate adenylyltransferase (EC 2.7.7.27).

ADP-glucose pyrophosphorylase has been isolated in homogeneous form from an Escherichia coli B mutant, AC70R1, derepressed in the synthesis of glycogen synthetic enzymes. The enzyme has been found to be identical with the wild type enzyme with respect to kinetic properties, molecular weight, and immunological reactivity. The AC70R1 enzyme is composed of four identical subunits of molecular weight of approximately 50,000. This is based on the findings that: (a) gel electrophoresis under denaturing conditions shows only one component; (b) tryptic mapping shows only enough peptides to account for a single polypeptide subunit; (c) digestion with carboxypeptidase B releases stoichiometric amounts of arginine; and (d) NH2-terminal sequencing shows a single sequence for the first 27 residues.

Adenosine Diphosphate Glucose↗