Kinetic sensitivity of a receptor-binding radiopharmaceutical: technetium-99m galactosyl-neoglycoalbumin.

Kinetic sensitivity is the ability of a physiochemical parameter to alter the time-activity curve of a radiotracer. The kinetic sensitivity of liver and blood time-activity data resulting from a single bolus injection of [99mTc]galactosyl-neoglycoalbumin [( Tc]NGA) into healthy pigs was examined. Three parameters, hepatic plasma flow scaled as flow per plasma volume, ligand-receptor affinity, and total receptor concentration, were tested using [Tc]NGA injections of various molar doses and affinities. Simultaneous measurements of plasma volume (iodine-125 human serum albumin dilution), and hepatic plasma flow (indocyanine green extraction) were performed during 12 [Tc]NGA studies. Paired data sets demonstrated differences (P(chi v2) less than 0.01) in liver and blood time-activity curves in response to changes in each of the tested parameters. We conclude that the [Tc]NGA radiopharmacokinetic system is therefore sensitive to hepatic plasma flow, ligand-receptor affinity, and receptor concentration. In vivo demonstration of kinetic sensitivity permits delineation of the physiologic parameters that determine the biodistribution of a radiopharmaceutical. This delineation is a prerequisite to a valid analytic assessment of receptor biochemistry via kinetic modeling.

On the cooperative binding of large ligands to a one-dimensional homogeneous lattice: the generalized three-state lattice model.

We present the general secular equation for three-state lattice models for the cooperative binding of large ligands to a one-dimensional lattice. In addition, a closed-form expression for the isotherm is also obtained, that can be used with all values of the cooperativity parameter omega(0 less than omega less than infinity) thus eliminating the need for multiple equations.

Evidence for multiple protein constituents of the somatostatin receptor in pituitary tumor cells: affinity cross-linking and molecular characterization.

Previous studies have shown that somatostatin receptors on AtT-20 and GH3 pituitary tumor cells show relative preference for binding somatostatin-28 (S-28) and somatostatin-14 (S-14), respectively. Here we have attempted to determine whether this selectivity can be explained by molecular heterogeneity of the receptor. Cells were incubated with [125I-Tyr11]S-14, [125I-Leu8-D-Trp22,Tyr25]S-28, and [125I-Tyr3]SMS, and the bound ligand was chemically cross-linked with bis-[2-succinimido-oxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate, or dithiobis (succinimidyl propionate). The solubilized cross-linked material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. [125I-Tyr11]S-14 labeled three specific receptor proteins of 57K, 42K, and 27K mol wt in AtT-20 cells. The relative proportions of the protein bands were unaltered by the use of whole cells or cell membranes or by the inclusion of dithiothreitol or antiproteolytic agents. With both [125I-Tyr11]S-14 and [125I-LTT]S-28, the 57K protein constituted the major labeled component, representing 70-75% of the total cross-linked proteins. Labeling of the three protein species by [125I-Tyr11]S-14 and [125I-LTT]S-28 was inhibited by both S-14 and S-28 in a dose-dependent manner. S-28 was 10-20 times more potent than S-14 for inhibiting the labeling by both ligands of the principal receptor species of 57K. By contrast, when a radioiodinated derivative of the octapeptide analog octreotide ([125I-Tyr3]SMS) was used as ligand, the 27K protein was preferentially labeled, whereas the 57K and 42K bands were detected only as minor components. Labeling of GH3 cells with [125I-Tyr11]S-14 and [125I-LTT]S-28 revealed three cross-linked proteins of 57K, 42K, and 27K mol wt similar to those observed in AtT-20 cells. However, in this cell line the 27K protein, not the 57K species, was the dominant component identified with these two ligands, comprising 40-50% of the total cross-linked proteins. These results suggest that there are three somatostatin receptor proteins of 57K, 42K, and 27K in pituitary cells. In AtT-20 cells, the 57K protein constitutes the major receptor protein labeled by [125I-Tyr11]S-14 and [125I-LTT]S-28, whereas the 27K protein is the major species labeled by [125I-Tyr3]SMS. The 27K, not the 57K, moiety is the principal receptor form in GH3 cells. Such ligand- and tissue-selective binding by the somatostatin receptor provides strong evidence for receptor molecular heterogeneity.

Lymphocyte recognition of psoriatic endothelium: evidence for a tissue-specific receptor/ligand interaction.

Epidermotropic lymphocytes are an essential cellular component of the skin-associated lymphoid tissues (SALT). Dermal lymphocytic infiltrates are also characteristics of inflammatory dermatoses, such as psoriasis, and may be involved in the pathogenesis of the disease, although the mechanisms by which lymphocytes are recruited into these sites are not known. We have used an in vitro lymphocyte adherence assay to test the hypothesis that specialized endothelial cells are present in inflamed skin, and are capable of supporting lymphocyte adherence and promoting lymphocyte emigration. In this assay, we assessed the binding of lymphocytes overlaid onto frozen sections of normal and psoriatic skin. Peripheral blood mononuclear cells (PBMC) from patients, from healthy volunteers, and from rat thoracic duct bound specifically to dermal endothelia in psoriatic plaques, in steroid-resistant areas of plaques, but not in uninvolved skin or skin from healthy individuals. Analysis of the binding properties of lymphocyte subsets revealed preferential adherence by CD4+ T cells as compared with CD8+ T cells and to B cells. Interestingly, PBMC from patients undergoing ultraviolet light therapy failed to adhere to autologous skin or to lesion-containing skin sections from untreated patients. Additional studies indicate that the lymphocyte-endothelial interaction is an energy- and calcium-dependent process and involves surface glycoprotein and carbohydrate moieties, requirements similar to those found in specific lymphocyte interactions with high endothelial venules in lymph nodes during the homing process. Pretreatment of lymphocytes with antibodies directed against homing receptors mediating migration into lymph nodes and into gut-associated lymphoid tissues, however, did not interfere with lymphocyte adherence to psoriatic endothelium. In contrast, anti-lymphocyte function associated antigen (LFA)-1 antibody partially inhibited lymphocyte binding to chronic plaques. We conclude that a tissue-specific receptor/ligand interaction independent of LFA-1 directs lymphocyte emigration from the dermal microvasculature into the psoriatic dermis and that LFA-1 plays only an accessory role in the adhesion process.

Characterization of the receptor for lymphotoxin; a spontaneous internalization without recycling and ligand-induced downregulation in HL-60 cells.

The receptor system for lymphotoxin (LT) was investigated by use of the human promyelocytic HL-60 cell line. Utilizing subcellular fractionation, internalization of cell surface bound recombinant LT (rLT) and transfer to lysosomes followed by degradation was demonstrated. Binding of rLT to its cell surface receptor induced a downregulation of the receptor which was persistent for at least 6 h. Downregulation of the receptors from the cell surface by activation of protein kinase C with the diacylglycerol OAG was completely reversible but the recovery of the binding capacity was dependent on protein synthesis as it was inhibited by cycloheximide. Treatment with cycloheximide alone resulted in a loss of binding capacity with a half life of approximately 2 h, suggesting a spontaneous consumption of receptors. Affinity cross-linking revealed three ligand-receptor complexes with Mr of 85, 105 and 125 kDa. Because of a strong tendency for ligand polymerization these results suggest oligomer binding of the ligand to a single receptor molecule with an apparent Mr of 70 kDa. N-linked glycosylation constituted 4 to 5 kDa of the total molecular weight. In conclusion, we demonstrated a spontaneous internalization of the receptor for LT without recycling, and that ligand binding resulted in an irreversible downregulation of the receptor.

Interleukin 2-like material in human epidermis: a ligand for the human interleukin 2-receptor 55 kD alpha chain.

The antirecombinant interleukin 2 (rec-IL-2) monoclonal antibody (moAb) 15.2 cross-reacts with a skin antigen located at the cell surface of human keratinocytes within the granular layer. This study extends the analysis of this IL-2-like material to its reactivity with eight antibodies raised against natural IL-2, rec-IL-2 or IL-2 peptides. Four among them were found to react with the granular epidermal layer as well as with a simian virus 40 (SV40) transformed human keratinocyte cell line. Each of these four antibodies gave similar labeling patterns, although with different intensities, and competitively inhibited each other. Analysis at the messenger RNA level in epidermal cells and SVK 14 also indicated that this material is very likely different from IL-2. From the knowledge, for some of these antibodies, of the amino-acid regions they recognize on the IL-2 molecule, it is inferred that the skin antigen shares with IL-2 at least two overlapping epitopes located in the 33-54 amino-acid region of IL-2, a region that has been shown to be involved in the binding of IL-2 to the IL-2-receptor (IL-2-R) 55 kD chain. Indeed, a purified recombinant soluble species of this IL-2-R is shown in this study to bind specifically to the IL-2-like skin material. As far as IL-2-R bearing cells are found in normal epidermis (Langerhans cells) and as important infiltrates of IL-2-R positive T lymphocytes are often encountered in cutaneous diseases, a potential role for this IL-2-like material in skin immunophysiopathology is suggested.

Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms.

The turnover of the colony-stimulating factor 1 receptor (CSF-1R), the c-fms proto-oncogene product, is accelerated by ligand binding or by activators of protein kinase C (PKC), such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The mechanisms of ligand- and TPA-induced downmodulation were shown to differ by the following criteria. First, in cells in which PKC was downmodulated, CSF-1R reexpressed at the cell surface remained sensitive to ligand but was refractory to TPA-induced degradation. Second, a kinase-defective receptor containing a methionine-for-lysine substitution at amino acid 616 at its ATP-binding site failed to undergo ligand-induced downmodulation but remained responsive to TPA. Following CSF-1 stimulation, no intermediates of receptor degradation could be immunoprecipitated with polyvalent antisera to CSF-1R. In contrast, TPA induced specific proteolytic cleavage of the receptor near its transmembrane segment, resulting in the release of the extracellular ligand-binding domain from the cell and the generation of an intracellular fragment containing the kinase domain. Two-dimensional phosphopeptide mapping demonstrated no new sites of phosphorylation in response to TPA in either the residual intact receptor or the intracellular proteolytic fragment. Therefore, PKC appears not to trigger downmodulation by directly phosphorylating the receptor but, rather, activates a protease which recognizes CSF-1R as a substrate.

Structure of tyrosyl-tRNA synthetase refined at 2.3 A resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate.

The crystal structure of tyrosyl-tRNA synthetase (EC 6.1.1.1) from Bacillus stearothermophilus has been refined to a crystallographic R-factor of 22.6% at 2.3 A resolution using a restrained least-squares procedure. In the final model the root-mean-square deviation from ideality for bond distances is 0.018 A and for angle distances is 0.044 A. Each monomer consists of three domains: an alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet, an alpha-helical domain (248 to 318) containing five helices, and a disordered C-terminal domain (319 to 418) for which the electron density is very weak and where it has not been possible to trace the polypeptide chain. Complexes of the enzyme with the catalytic intermediate tyrosyl adenylate and the inhibitor tyrosinyl adenylate have also been refined to R-factors of 23.9% at 2.8 A resolution and 21.0% at 2.7 A resolution, respectively. Formation of the complexes results in some crystal cracking, but there is no significant difference in the conformation of the polypeptide chain of the three structures described here. The relative orientation of the alpha/beta and alpha-helical domains is similar to that previously observed for the "A" subunit of a deletion mutant lacking the C-terminal domain. Differences between these structures are confined to surface loops that are involved in crystal packing. Tyrosyl adenylate and tyrosinyl adenylate bind in similar conformations within a deep cleft in the alpha/beta domain. The tyrosine moiety is in the equivalent position to that occupied by tyrosine in crystals of the truncated mutant and makes similar strong polar interactions with the enzyme. The alpha-phosphate group interacts with the main-chain nitrogen of Asp38. The two hydroxyl groups of the ribose form strong interactions with the protein. The 2'-hydroxyl group interacts with the carboxylate of Asp194 and the main-chain nitrogen of Gly192 while the 3'-hydroxyl interacts with a tightly bound water molecule (Wat326). The adenine moiety appears to make no significant polar interactions with the protein. The results of site-directed mutagenesis studies are examined in the light of these refined structures.

Modification of ligand binding to the Na+/K+-activated ATPase.

Interactions between the ligands Mg2+, K+, and substrate and the Na+/K+-activated ATPase were examined in terms of a rapid-equilibrium, random-order, terreactant kinetic scheme for the K+-nitrophenyl phosphatase reaction that is catalyzed by this enzyme. At 37 degrees C and pH 7.5 the derived values for the dissociation constants from the free enzyme were 0.2, 0.08, and 1.4 mM for Mg2+, K+, and substrate, respectively. For Mg2+ interactions, the presence of 20% (v/v) dimethyl sulfoxide (Me2SO) increased the calculated affinity 25-fold; higher concentrations increased affinity still further. Neither reducing the temperature to 20 degrees C nor altering the pH from 6.5 to 8.3 appreciably changed the affinity for Mg2+ in the absence or presence of Me2SO. The Mg2+ sites are thus characterized by an absence of functional groups ionizable in the pH range 6.5-8.3, with binding driven by entropy changes, and with Me2SO, probably through solvation effects on the protein, increasing affinity for Mg2+ close to that for Ca2+ and Mn2+. By contrast, for K+ interactions, the presence of 20% Me2SO increased the calculated affinity only by half; moreover, reducing the temperature to 20 degrees C and the pH to 6.5 both increased affinity and diminished the response to Me2SO. The K+ sites are thus characterized by a marked sensitivity to pH and temperature, presumably through alterations in enzyme conformational equilibria that in turn are modifiable by Me2SO. Inhibition by higher concentrations of Mg2+, which varies inversely with the K+ concentration, was decreased by Me2SO. Finally, for substrate interactions, the presence of 20% Me2SO increased the calculated affinity 4-fold, and, as for Mg2+-binding, neither reducing the temperature nor varying the pH over the range 6.5-8.3 appreciably altered the affinity in the absence or presence of Me2SO. Thus, the substrate sites, like the Mg2+ sites, are characterized by an absence of functional groups ionizable in this range, with binding driven by entropy changes, and with Me2SO increasing affinity for substrate, in this case probably through favoring the partitioning of substrate from the medium into the hydrophobic active site.

Evaluation of a semiautomatic cell harvester filtration for the determination of beta-adrenoceptors in human mononuclear leukocytes.

The ligand affinity (Kd) and the number (Bmax) of beta-adrenoceptors in human mononuclear leukocytes was measured using a semiautomatic cell harvester method for the separation of bound and free ligand. The method was first evaluated in terms of contamination, nonspecific filter binding, and well-to-well reproducibility. A comparison was then made with the conventional manual filtration method. The Kd values for the binding of [125lodo]-cyanopindolol to mononuclear leukocyte membranes were independent of the method, whereas the Bmax values were systematically higher (around 30%) when determined with the cell harvester filtration method. A highly significant correlation was found between the Bmax values obtained with both methods.

1H-NMR and protection studies of interactions between ligands and bovine pancreatic phospholipase A2.

A number of long-chain amines and naphthylamine sulfonates have been studied for their ability to inhibit bovine pancreatic phospholipase A2 (PLA2) and to protect PLA2 against alkylation of the active site histidine by p-bromophenacyl bromide. Their areas of interaction on the enzyme were further delineated using observations of chemical shift changes of assigned aromatic signals in the 1H-NMR spectrum of PLA2, while the bound conformations of two amine inhibitors were revealed using transferred nuclear Overhauser effects. The alkyl amines bind rather non-specifically on the surface of the enzyme, over the active site cleft and the interface recognition site.

Localization and quantification of hormones, ligands, and mRNA with affinity-gold probes.

The application of immunogold techniques to localize pituitary hormones produces label that can be quantified and correlated with different secretory states. This report focuses on three major applications of the technology. In the first set of studies, immunogold labels for adrenocorticotropin (ACTH) or luteinizing hormone (LH beta) and follicle-stimulating hormone (FSH beta) were applied to ultrathin sections of pituitaries from adrenalectomized rats or from rats in different stages of the estrous cycle. During the first week after adrenalectomy, ACTH cell area increased. The concentration of immunoperoxidase label (amount of label/area of the corticotropes) decreased. Counts of gold markers showed that there were no changes in the concentration of antigens per granule. Three weeks after adrenalectomy, the amount of immunoperoxidase label increased along with the concentration of that label. The concentration of gold label for ACTH on granules also increased. All changes correlated well with increases in serum ACTH stimulated by adrenalectomy. In the studies of cycling rats, gonadotropes showed increases in the number of gold markers for LH beta or FSH beta per granule area just before an elevation in serum levels. There were also increases in the proportion of granules that contained only LH beta or FSH beta (monohormonal) before the rise in secretion. Thus, nonparallel release of gonadotropins might be attributed to changes in the ratio of gonadotropins packaged per granule. In the second series of studies, avidin-gold labels were used to identify sites of binding of biotinylated ligands. These studies illustrate and quantify binding by biotinylated gonadotropin-releasing hormone (GnRH) to ovarian or pituitary target cells. Triple-labeling protocols (avidin-peroxidase followed by immunogold) show that the target cells in the pituitary contain gonadotropins. In the third set of studies, avidin gold or avidin peroxidase was used to label sites of hybridization of a biotinylated cRNA probe to gonadotropin beta subunit mRNA. The sites of hybridization appear on rough endoplasmic reticulum; however, further work is needed to improve cell ultrastructure and perserve antigens. Triple-labeling protocols (avidin-peroxidase followed by immunogold) show the feasibility of the technique as well as the need for further refinement. To summarize, these studies describe multiple applications of gold labels for the localization of antigens, ligands, and mRNA. The labels are sensitive for detection of antigens and ligands and easily quantified. Quantitative analyses show changes in concentration of gold label that correlate well with secretory states.

Immunogold-surface replica study of ADP-induced ligand binding and fibrinogen receptor clustering in human platelets.

Platelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP-activated human platelets. We found that the gold prove was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa binding domains of fibrinogen--namely, the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.

Structural basis of beta-adrenergic receptor function.

Receptors that mediate their actions by stimulating guanine nucleotide binding regulatory proteins (G proteins) share structural as well as functional similarities. The structural motif characteristic of receptors of this class includes seven hydrophobic putative transmembrane domains linked by hydrophilic loops. Genetic analysis of the beta-adrenergic receptor (beta AR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein. On the basis of these studies, a model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Other interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the beta AR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphiphilic alpha helix. This region of the beta AR is also critical for sequestration, which accompanies agonist-mediated desensitization, to occur. Structural similarities among G protein-linked receptors suggest that the information gained from the genetic analysis of the beta AR should help define functionally important regions of other receptors of this class.

Experimental design optimisation: theory and application to estimation of receptor model parameters using dynamic positron emission tomography.

The general framework and various criteria for experimental design optimisation are presented. The methodology is applied to the estimation of receptor-ligand reaction model parameters with dynamic positron emission tomography data. The possibility of improving parameter estimation using a new experimental design combining an injection of the beta+-labelled ligand and an injection of the cold ligand is investigated. Numerical simulations predict a remarkable improvement in the accuracy of the parameter estimates with this new experimental design and particularly the possibility of separate estimations of the association constant (k+1) and of the receptor density (B'max) in a single experiment. Simulation predictions are validated using experimental PET data in which parameter uncertainties are reduced by factors ranging from 17 to 1000.

From analysis to synthesis: new ligand binding sites on the lactate dehydrogenase framework. Part I.

In Part I of this article, the naturally evolved protein framework of lactate dehydrogenase is investigated by genetically introduced modifications which reveal the structural basis of its catalytic and substrate-binding properties. In Part II (to be published in the April issue of TIBS), this analytical information is exploited in the design of two modified forms of the enzyme; one which is specific for a new substrate and one which lacks allosteric regulation.

Absence of peripheral-type benzodiazepine binding sites in renal carcinoma: a potential biochemical marker.

In an attempt to identify a tumour marker, we investigated peripheral-type benzodiazepine binding sites (PBS) in kidney specimens obtained from patients who underwent nephrectomy due to a renal mass. [3H]PK 11195, an isoquinoline carboxamide derivative, was used as a ligand. Binding assays were conducted on samples of membrane homogenate taken from both the healthy portion and the tumour site of the kidney. It was found that binding characteristics of benign tumours and normal kidney tissues were not significantly different, i.e. equilibrium dissociation constants of 2.20 +/- 0.73 and 2.38 +/- 0.98 nM, respectively, and maximal number of binding sites of 3190 +/- 1081 and 4189 +/- 998 fmol/mg protein, respectively. In contrast, no PBS were detectable in renal carcinoma. The absence of PBS in malignant renal tissues may serve as a biochemical marker for tumours of the kidney.

Region-specific antiglucocorticoid receptor antibodies selectively recognize the activated form of the ligand-occupied receptor and inhibit the binding of activated complexes to deoxyribonucleic acid.

A synthetic 18-amino acid peptide (Cys500-Lys517) was used to raise polyclonal antibodies in rabbits to the glucocorticoid receptor (GR). The sequence of this peptide is identical to that of residues 500-517 of the rat and 481-498 of the human GR. This sequence overlaps the carboxy-terminal end of the core DNA-binding domain and the amino-terminus of the hinge region of the receptor. Antiserum (AP64) was obtained which recognized both human and rat GR, as determined by immunoblots of receptors immunopurified with authentic anti-GR antibodies, immunoadsorption of both specific [3H]dexamethasone-bound GR and 98K receptors that were specifically covalently labeled by [3H]dexamethasone mesylate, and AP64-induced shifts in the elution position of monomeric [3H]dexamethasone-bound GR from Sephacryl S-300. The specificity of AP64 was demonstrated by the ability of the immunizing peptide, but not a peptide of similar length, to inhibit both the antibody-induced change in elution position from Sephacryl S-300 and the antibody-mediated immobilization of [3H]dexamethasone-bound complexes by protein-A. Further studies indicated that AP64 did not react with native steroid-free GR or with steroidbound (or affinity-labeled) unactivated GR, but did selectively associated with monomeric activated, steroid-bound (or affinity labeled) complexes. AP64 also inhibited the DNA binding of activated complexes in a manner that was specifically blocked by the immunizing peptide. Collectively, these data allow the direct localization of a structural region of the GR that is occluded in the unactivated complex but exposed as a result of activation.

Human microsomal glutathione S-transferase. Its involvement in the conjugation of hexachlorobuta-1,3-diene with glutathione.

A microsomal glutathione S-transferase (GST) was purified from human liver. This enzyme was shown to have characteristics similar to those of the rat microsomal GST described by Morgenstern & De Pierre [(1983) Eur. J. Biochem. 134, 591-597]. The specific activity of human microsomal GST towards 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide can be stimulated by treating the enzyme with N-ethylmaleimide. This enhancement of activity is accompanied by increased sensitivity to inhibition by haematin and cholic acid. The subunit Mr values of the rat and human enzymes are similar (approx. 17,300), and the proteins are immunologically related. During purification, both human and rat microsomal GST enzymes are the only hepatic proteins obtained from Triton X-100-solubilized microsomal fractions that show activity towards the nephrotoxin hexachlorobuta-1,3-diene. The involvement of microsomal GST in toxification reactions is discussed.

Ligand-independent tyrosine phosphorylation of EGF receptor and the erbB-2/neu proto-oncogene product is induced by hyperosmotic shock.

Treatment of intact cells with media containing high concentrations of ionic and non-ionic solutes induced increased tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and the protein product of the erbB-2/neu proto-oncogene. This self phosphorylation occurred in the absence of EGF or other growth factors. High concentrations of solutes did not activate phosphorylation of either isolated EGF receptor or EGF receptor solubilized by non-ionic detergents. No evidence for receptor dimerization was found in response to hyperosmotic shock. Since receptor dimers have been implicated in the EGF-induced activation of EGF receptor, hyperosmotic shock may activate EGF receptor by a different mechanism.