Differences between cytosol receptor complexes with corticosterone and dexamethasone in hippocampal tissue from rat brain.

The binding of [3H]corticosterone and [3H]dexamethasone to soluble macromolecules in cytosol of the hippocampal region of the brain has been studied in adrenalectomized male rats. Unlabeled dexamethasone appears to be a less effective competitor than corticosterone in the binding of [3H]corticosterone, while both unlabeled steroids compete equally well for the binding or [3H]dexamethasone. Further investigation of macromolecular complexes with [3H]dexamethasone and [3H]corticosterone revealed that they differ from each other in their behavior during ammonium sulfate precipitation, BioRad A-5M gel permeation chromatography, DE-52 anion exchange chromatography and DNA-cellulose chromatography. (1) After exposure to a 33% ammonium sulfate solution relatively more [3H]dexamethasone complex than [3H]corticosterone complex is precipitated. (2) Treatment of the cytosol with 0.3 M KCl gives disaggregation of the supramolecular 3H-labeled corticoid complexes which are seen eluting with the void volume during gel permeation chromatography on Biorad A-5M at low ionic strength. In 0.3 M KCl, the [3H]dexamethasone complex has an elution volume somewhat smaller than that of bovine serum albumin, while the [3H]-corticosterone complex in 0.3 M KCl is too unstable to survive chromatography with A-5M. (3) Chromatography on DE-52 resolved the 3H-labeled corticoid complexes into three binding components. The complex with [3H]dexamethasone contains a higher percentage (85%) of a component less firmly attached (i.e. eluted by 0.15 M KCl) to the anion exchange resin than is observed for the complex with [3H]corticosterone (49%). (4) The complexes with 3H-labeled corticoids display an enhanced affinity for calf thymus DNA adsorbed to cellulose following "activation", warming to 25 degrees C for 15 min. Concurrently, a fraction of the [3H]dexamethasone complex becomes able to more firmly attach to the DE-52 anion exchange resin. These results with the binding of the cytosol hormone-receptor complexes to DNA-cellulose do not explain the marked in vivo preference of hippocampus for the cell nuclear uptake of [3H] corticosterone. However, the other differences in the properties of the complexes formed with the two labeled glucocorticoids support our previous inference that there may be more than one population of adrenal steroid "receptors" in brain tissue.     

Interactions of rab5 with cytosolic proteins.

Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.     

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.     

Effect of endogenous corticosterone on the determination of dexamethasone receptor levels in rat liver cytosol

The effect of endogenous corticosterone on the quantitative measurement of dexamethasone receptors in liver cytosols from developing rats has been studied. Liver cytosols from adrenalectomized rats were preincubated with increasing concentrations of nonlabeled corticosterone and the levels of detectable dexamethasone receptors were subsequently determined either directly or after removal of unbound corticosterone. Corticosterone concentrations of 50 nM or lower had no significant effect on the specific binding of labeled dexamethasone. Higher concentrations of corticosterone resulted in under-estimation of dexamethasone receptor levels. The mean levels of endogenous corticosterone in liver cytosols from 19.5- to 21.5- day fetuses, 22-day fetuses, 6-day-old immature rats and adult rats were 27.40, 11.91, 0.81 and 4.05 nM, respectively. It is concluded that variations in the levels of circulating corticosterone in the rat under normal physiological conditions have no significant effect on the quantitative measurement of total (occupied and unoccupied) receptor sites for dexamethasone in liver cytosol. This is supported by the finding that prior treatment of liver cytosols, from rats at different stages of development, with charcoal to remove unbound steroids has no effect on the amount of detectable dexamethasone receptors.     

Interactions of basic polypeptides and proteins with calmodulin.

Low concentrations (less than 10 microgram/ml) of a number of highly basic polypeptides inhibit the calmodulin-stimulated cyclic nucleotide phosphodiesterase. Inhibitory compounds include synthetic polypeptides [polylysine (D and L) and polyarginine] and basic proteins (protamine, histones H1, H2A, H2B, H3 and H4 and myelin basic protein). Polylysine of mol.wt. about 2000 or higher was inhibitory, but pentalysine did not inhibit. Other basic proteins and compounds did not inhibit, including bradykinin, spermine and putrescine. In mixtures of calmodulin and basic protein, complexes were formed whether Ca2+ was present or not. This was true for polylysine, myelin basic protein and histone H2B. These interactions suggest that the inhibition of the phosphodiesterase is due to interaction of these basic proteins with calmodulin. The wide variety of basic polypeptides and proteins that affect the calmodulin stimulation of phosphodiesterase indicates that these interactions are not specific.     

Haem-binding proteins of the rat liver cytosol.

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino[3H]laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [3H]haem-labelled mitochondria, [3H]haem-labelled microsomes or with [3H]haemin. These results are discussed with particular reference to ligandin.     

Two-dimensional electrophoresis of membrane proteins. Factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI greater than 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112-120) gives good resolution of proteins of pI 5-8, including cytochromes P-450, but fails to resolve a major acidic protein of pI less than 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R . N+(CH3)2 . CH2CH2CH2SO3- are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C14 or C16) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Interactions of bacterial lipopolysaccharide with microtubule proteins.

Bacterial LPS is a potent stimulator of immune cells, but its mechanisms are unknown. A possible role for microtubules in LPS actions has been indicated by previous findings that the microtubule-active agent, taxol, can mimic some effects of LPS in macrophages from normal strains of mice, but not from genetically LPS-hyporesponsive strains. In this report we demonstrate that isolated microtubules from mouse brain can bind LPS in vitro. LPS and tubulin coeluted through a gel filtration column, and LPS was cross-linked to microtubule proteins with an iodinatable, photoreactive agent, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3'-dithiopropionate. beta-Tubulin and microtubule-associated protein-2 (MAP), a predominant MAP in the brain, bound LPS specifically. Cross-linking was inhibited by an excess of unlabeled LPS or partially by unlabeled lipid A, but not by 2 M NaCl. Under the same conditions, neither myosin nor soybean trypsin inhibitor was labeled by the photoaffinity LPS probe, nor did these proteins compete for binding of LPS to beta-tubulin. These findings support the hypothesis that the microtubule network could be an intracellular target for LPS, and suggest further that a beta-tubulin-associated MAP could have an important role in LPS actions.     

Interactions of cytosol enzymes with the red blood cell membrane

It is not dubious that the regulation of erythrocyte metabolism occurs across the membrane and that accordingly interactions between cytosolic enzymes and membrane components necessarily exist. Several aspects of such relationships were reviewed. The results of some experiments carried out in non-physiological conditions should be carefully interpreted. However it can be accepted that some enzymes undergo reciprocal translocations between cytosol and membrane and that very probably these transfers play a role in the control of metabolic regulation in the red cell.     

Interactions of proteins and cholesterol with lipids in bilayer membranes.

Mixtures of lipids and protein, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-PO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, Tt, of the lipid and aggregated below Tt. For mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains were shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present. In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50-200 nm in length, around smooth patches of lipid. Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperature of the lipid is discussed.     

Cyclic AMP-binding proteins and protamine kinases in porcine thyroid cytosol.

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.     

Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.

Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.     

The rat-liver microsomal AP endonuclease. The endoplasmic reticulum is presented as a net thrown into the cytosol to capture newly synthesized karyophilic proteins

Although most of the rat-liver AP (apurinic/apyrimidinic) endonuclease is in chromatin, some activity is found in microsomes. A quantitative assay of the microsomal AP endonuclease is described. The enzyme is a peripheral membrane protein that is located on the outside surface of microsomes. All the binding sites on the microsomes appear to have the same affinity for the AP endonuclease, suggesting the presence of receptors for the enzyme. The AP endonuclease is displaced from its membrane attachment by submicromolar concentrations of the karyophilic signal of SV-40 T antigen. The AP endonuclease receptors are likely to be on the cytosolic side of the endoplasmic reticulum. It is suggested that binding of the protein to these receptors might be the first step of the transport mechanism that enables the AP endonuclease to penetrate into the nucleus. The same mechanism utilizing the same receptors might be used by other karyophilic proteins, including SV-40 T antigen.     

Differential effects of corticosterone and dexamethasone on hippocampal neurogenesis in vitro

Prenatal stress during fetal development results in the blockade of neurogenesis in the dentate gyrus in adulthood. Present study was undertaken to investigate the dominant role of the glucocorticoid receptors in corticosterone actions on the neurogenesis of fetal hippocampal progenitor cells. For that purpose, expressions of key molecules affected by corticosterone and dexamethasone were compared during proliferation and differentiation of the hippocampal progenitor cells. Corticosterone (2 microM) significantly decreased the number of bromodeoxyuridine-labeled cells (about 50%) and caused the dendritic atrophy in microtubule-associated protein 2-labeled cells. The expressions of NeuroD, BDNF, and NR1 mRNA levels and protein levels of p-ERK and p-CREB were remarkably decreased by corticosterone in a dose-dependent manner. In contrast, dexamethasone, a glucocorticoid receptor (GR) specific agonist, had an inhibitory effect on proliferation, but not differentiation. It is concluded that corticosterone elicits its effects on neurogenesis including proliferation and differentiation whereas stimulation of the glucocorticoid receptor is sufficient to decrease only proliferation.     

Phosphorylation of brain cytosol proteins. Effects of phospholipids and calmodulin

Calmodulin was removed from brain cytosol by DEAE-52 chromatography or by affinity chromatography employing fluphenazine-Sepharose. The substrates phosphorylated by endogenous protein kinase after chromatography differed depending on the method used, and both chromatographic methods altered the phosphorylation pattern as compared to untreated cytosol. Cytosol, chromatographed on fluphenazine-Sepharose, retained most of the characteristics of untreated cytosol. Both calmodulin and phospholipids increased the phosphorylation of specific but separate brain cytosol proteins in a Ca2+-dependent manner. The effects of phospholipids could be mimicked by the detergent, sodium dodecyl sulfate, and the hydrophobic probe, 8-anilino-1-naphthalenesulfonate. Furthermore, the calmodulin-induced increase in phosphorylation, but not that produced by phospholipids, was blocked by 8-anilino-1-naphthalenesulfonate. These results suggest that the effects of phospholipids may not be due to the presence of a specific phospholipid-sensitive protein kinase in cytosol, but rather to a general interaction of hydrophobic probes with either specific substrate proteins or with the Ca2+-calmodulin-dependent protein kinase itself.