Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Solubilization of the microsomal fraction from bovine kidney by Triton X-100 or by 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulfonate (CHAPS) increased 2-fold the thermodynamic association constant for hGH. While solubilization with CHAPS did not change the 13-fold preferential binding of human growth hormone (hGH) over ovine prolactin (oPRL), solubilization with Triton X-100 increased this preference to 47-fold. The binding was optimal at pH 7-7.5 in the presence of 10 mM of MgCl2 or CaCl2. The association rate with hGH was identical in the microsomal and Triton X-100 solubilized fractions but the dissociation was slower in the latter. Only partial dissociation was observed at neutral pH. Full dissociation was, however, achieved by lowering the pH to 4-5, indicating that the binding was not covalent. Gel filtration studies of the Triton X-100 solubilized fraction after preincubation in the presence of reducing agent revealed two sharp peaks of activity, one having Mr of greater than 700 kDa that represented the aggregated receptor, and the second, with Mr 110-115 kDa. The specificity of the partially purified receptors clearly shows that they are lactogenic and not somatogenic. They resemble lactogenic receptors found in other bovine organs, but differ from other species particularly in their differential affinities of PRL and hGH.
Mol Cell Endocrinol 1989 Jan
PMID:Solubilization and characterization of lactogenic hormone receptor from kidney of lactating cow. 274 17

The formation of heteroduplexes from linear duplex DNA, where one molecule possesses a DNA double-strand break, was assayed by agarose gel electrophoresis. Using unlabeled whole-length linear duplex DNA and 3H-labeled half-length linear duplex DNA (obtained from plasmid pACYC184), the appearance of 3H-labeled DNA that migrated as whole-length linear DNA was taken as evidence for formation of heteroduplex DNA. When the DNA mixtures were incubated with RecA, RecBCD, or Ssb proteins, or any double or triple combination of these proteins under a variety of reaction conditions, no heteroduplex DNA was detected. However, heteroduplex DNA was detected when the DNA mixtures were first incubated briefly with the RecBCD and Ssb proteins under reaction conditions that allow unwinding to proceed, and then the MgCl2 concentration was raised such that renaturation could proceed. The inclusion of the RecBCD and Ssb proteins was sufficient to catalyze the slow formation of heteroduplex DNA, but the presence of RecA protein greatly increased the kinetics. The roles of the RecBCD, Ssb and RecA proteins in heteroduplex formation in vitro are discussed.
Mol Gen Genet 1989 Apr
PMID:The roles of RecBCD, Ssb and RecA proteins in the formation of heteroduplexes from linear-duplex DNA in vitro. 274 19

Malate dehydrogenase from the extreme halophile, Halobacterium marismortui, is stable only in highly concentrated solutions of certain salts. Previous work has established that its physiological environment is saturated in KCl; it remains soluble is saturated NaCl or KCl solutions; also it unfolds in solutions containing less than 2.5 M-NaCl or -KCl, salt concentrations which are still relatively high. New data show that the structure of this enzyme can be stabilized in a range of high concentrations of Mg2+ or other "salting-in" ions, also with exceptional protein-solvent interactions. "Salting-in" ions, contrary to stabilizing protein structure, usually favour unfolding. These, and most other results concerning the structure, stability and solvent interactions of the protein cannot be understood in terms of the usual effects of salts on protein structure. In this paper, a novel stabilization model is proposed for halophilic malate dehydrogenase that can account for all observations so far. The model results from experiments on the protein in salt solutions chosen for their different effects on protein stability (potassium phosphate, a strongly "salting-out" agent, and MgCl2, which is "salting-in"), and previously published data from NaCl and KCl solutions (mildly "salting-out"). Enzymic activity and stability measurements were combined with neutron scattering, ultracentrifugation and quasi-elastic light-scattering experiments. The analysis showed that the structure of the protein in solution as well as the dominant stabilization mechanisms were different in different salt solutions in which this enzyme is active. Thus, in molar concentrations of phosphate ions, stabilization and hydration are similar to those of non-halophilic soluble proteins, in which the hydrophobic effect dominates. In high concentrations of KCl, NaCl or MgCl2, on the other hand, solution particles are formed in which the protein dimer interacts with large numbers of salt and water molecules (the mass of solvent molecules involved depends on the nature of the salt but it is approximately equivalent to the protein mass). It is proposed that, under these conditions, the hydrophobicity of the protein core is too weak to stabilize the folded structure and the main stabilization mechanism is the formation of co-operative hydrate bonds between the protein and hydrated salt ions. Model predictions are in agreement with all experimental results, such as the different numbers of solvent molecules found in the solution particles formed with different salts, the loss of the exceptional solvent interactions concomitant with unfolding at non-physiological salt concentrations, and the different temperature denaturation curves observed for different salt solutions.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1989 Aug 05
PMID:Stabilization of halophilic malate dehydrogenase. 279 58

The determination and characterization of a cannabinoid receptor from brain are reported. A biologically active bicyclic cannabinoid analgetic CP-55,940 was tritium-labeled to high specific activity. Conditions for binding to rat brain P2 membranes and synaptosomes were established. The pH optimum was between 7 and 8, and specific binding could be eliminated by heating the membranes to 60 degrees. Binding to the P2 membranes was linear within the range of 10 to 50 micrograms of protein/ml. Specific binding (defined as total binding displaced by 1 microM delta 9-tetrahydrocannabinol (delta 9-THC) or 100 nM desacetyllevonantradol) was saturable. The Kd determined from Scatchard analysis was 133 pM, and the Bmax for rat cortical P2 membranes was 1.85 pmol/mg of protein. The Hill coefficient for [3H]CP-55,940 approximated 1, indicating that, under the conditions of assay, a single class of binding sites was determined that did not exhibit cooperativity. The binding was rapid (kon approximately 2.6 x 10(-4) pM-1 min-1) and reversible (Koff approximately 0.016 min-1) and (koff' greater than 0.06 min-1). The two Kd values estimated from the kinetic constants approximately 55 pM and exceeded 200 pM, respectively. The binding of the agonist ligand [3H]CP-55,940 was decreased by the nonhydrolyzable GTP analog guanylylimidodiphosphate. The guanine nucleotide induced a more rapid dissociation of the ligand from the binding site, consistent with an allosteric regulation of the putative receptor by a G protein. The binding was also sensitive to MgCl2 and CaCl2. Binding of [3H]CP-55,940 was displaced by cannabinoid drugs in the following order of potency: CP-55,940 greater than or equal to desacetyllevonantradol greater than 11-OH-delta 9-THC = delta 9-THC greater than cannabinol. Cannabidiol and cannabigerol displaced [3H]CP-55,940 by less than 50% at 1 microM concentrations. The (-)-isomer of CP-55,940 displaced with 50-fold greater potency than the (+)-isomer. This pharmacology is comparable to both the inhibition of adenylate cyclase in vitro and the analgetic activity of these compounds in vivo. The criteria for a high affinity, stereoselective, pharmacologically distinct cannabinoid receptor in brain tissue have been fulfilled.
Mol Pharmacol 1988 Nov
PMID:Determination and characterization of a cannabinoid receptor in rat brain. 284 84

A modified procedure in two versions (micro, for 10 ml of phage lysate, and macro, for 200-500 ml) is described for preparing lambda phage DNA. The main advantage of the modified method is that it gives a possibility to isolate high-quality DNA from lambda phage lysates in 2-3 hrs. Only standard solutions (TE, NaCl, SDS, MgCl2, EDTA, RNAse A) were used throughout the whole protocol. Incubation with DNAse I and proteinase K was omitted and in microvariant concentration of the phage by PEG 6000 was excluded. Digestion by RNAse A was performed in solution with EDTA and SDS and leads to RNA degradation. The yields of DNA (0.5-2 micrograms per ml of L-broth) are similar to those obtained by other methods. DNA quality is better than in the samples of DNA prepared by other express-methods and practically the same as after CsCl centrifugation. DNA can be used for splitting by restriction enzymes, cloning and gene library construction.
Mol Biol (Mosk)
PMID:[Rapid isolation of phage lambda DNA]. 285 52

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.
Mol Cell Biol 1988 Aug
PMID:A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth. 290 23

Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.
J Mol Biol 1989 Jan 05
PMID:Crystallization and preliminary X-ray study of horse pancreatic lipase. 292 6

The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used. When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed. Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 degrees C) and magnesium chloride concentrations of 1 mM or 4 mM, a time-dependent inhibition of activity is observed. These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper. However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 degrees C) and magnesium chloride concentrations of 4 mM or 10 mM, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA. A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein. The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(+/- 1) nucleotides per RecA protein monomer at 4 mM-MgCl2 and 37 degrees C. Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last. In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mM-MgCl2. These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions. The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1987 Jan 05
PMID:Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein. Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA. 295 3

The statistical segment length of duplex DNA was determined in phage T4 ligase (poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMP forming), EC 6.5.1.1) buffer (50 mM-Tris . HCl (pH 7.8), 20 mM-dithiothreitol, 10 mM-MgCl2, 1 mM-ATP) at 12 degrees C to be 1030(+/- 116) A. This result was obtained by electron microscopic examination of the molecular distributions generated by T4 ligase-mediated joining of EcoRI-cleaved pBR322 DNA. This value of the statistical segment length was utilized in an extension of the Jacobson-Stockmayer theory on the probability of intramolecular cyclization in order to optimize DNA joining reactions that are of great utility in recombinant DNA experiments. Five cloning systems were analyzed: circular plasmid vectors that had been linearized with one or two restriction endonucleases, circular plasmids that had been tailed with deoxyhomopolymers before joining, lambda-type cloning vectors and cosmids. The results are tabulated for convenient use in molecular cloning experiments.
J Mol Biol 1985 Jan 20
PMID:Analysis and optimization of recombinant DNA joining reactions. 298 33

Rat hepatic prolactin receptor is regulated by sex steroids. A high level of the receptor was found in female rats but the level was nearly undetectable in males. Gonadectomy reduced the receptor level in females but increased the level in males. Administration of estradiol benzoate (0.05 mumoles/kg on alternate days subcutaneously for 9 days) to adult gonadectomized females increased the receptor level by 473% whereas the same treatment in adult gonadectomized males produced a more modest 276% increase. This sexually dimorphic pattern in the responsiveness to estrogen stimulation in adult rats appeared to be determined neonatally. Neonatal gonadectomy of male rats changed the hepatic response system to a more female pattern in adulthood. Replacement of testosterone (1.45 mumoles at days 1 and 3 after birth) to these neonatally gonadectomized male rats restored the male pattern. Diethylstilbestrol replacement (1.45 mumoles at days 1 and 3 after birth) to the neonatally gonadectomized male rats showed the same effect as neonatally administered testosterone. Scatchard analysis revealed that the observed changes in binding are related to changes in binding capacity but not affinity. Desaturation by 4 M MgCl2 indicated that the amount of endogenously bound hormone was negligible in our membrane preparations.
Mol Cell Endocrinol 1985 May
PMID:Prolactin receptor in rat liver: sex difference in estrogenic stimulation and imprinting of the responsiveness to estrogen by neonatal androgen in male rats. 298 35


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