Synthesis Of Binding Protein Research Articles

Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE+ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a supE-strain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel beta-strands organized into two orthogonally situated beta-sheets. The overall conformation of the protein resembles that of a clam--hence the term beta-clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg106 and Gln115. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the beta-clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations of several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Acinar heterogeneity of fatty acid binding protein expression in the livers of male, female and clofibrate-treated rats.

Liver fatty acid binding protein may play a role in the intracellular transport and compartmentation of long-chain fatty acid metabolism. The distribution of liver fatty acid binding protein in the hepatic acinus was determined by means of immunocytochemistry as well as by measurement of liver fatty acid binding protein in cellular protein selectively released from zone 1 and zone 3 cells by means of anterograde and retrograde liver perfusion with digitonin. In untreated male rats, specific immunocytochemical staining for liver fatty acid binding protein showed a declining portal-to-central hepatocellular gradient in intensity, consistent with the portal-to-central ratio of liver fatty acid binding protein abundance measured in effluents from digitonin-perfused livers of 1.6:1. Female and clofibrate-treated male rats, in both of which hepatic synthesis and abundance of liver fatty acid binding protein are greater than in untreated males, differed as well in the pattern of acinar expression of this protein. In females, periportal concentrations of liver fatty acid binding protein determined from the effluent of livers perfused anterograde with digitonin were similar to male values, whereas liver fatty acid binding protein concentration in pericentral hepatocytes determined from the effluent of retrograde perfused livers was increased, resulting in a marked attenuation of the portal-to-central gradient of this protein; this was also apparent on immunocytochemistry. Clofibrate-treated rats, in contrast, displayed a panacinar increase in liver fatty acid binding protein with maintenance of the portal-to-central ratio observed in untreated males. We conclude that there exists a declining portal-to-central gradient in liver fatty acid binding protein cellular abundance in the hepatic acinus of untreated male rats. Furthermore, the increased synthesis and abundance of liver fatty acid binding protein in female and clofibrate-treated male rats results in two different alterations in the acinar expression of this protein, i.e. a pericentral increase (female) or a panlobular increase (clofibrate). Elucidation of the relationship between the zonation of hepatic fatty acid metabolism and the acinar expression of liver fatty acid binding protein should provide a more detailed understanding of the function of this protein.

Estro-progestogen combinations: Effect on sex Steroid Binding Protein (SBP) synthesis

Estro-progestogen combinations: Effect on sex Steroid Binding Protein (SBP) synthesis

The relative rate of synthesis and levels of single-stranded DNA binding protein during induction of SOS repair in Escherichia coli.

Induction of the SOS response in Escherichia coli results in an increase in the relative rate of synthesis of single-stranded DNA binding protein (SSB). In contrast to RecA protein, this increase is slow and does not lead to higher SSB levels. The significance of ssb induction to SOS repair is discussed.

Synthesis and secretion of juvenile hormone binding protein by fat body from the cockroach Leucophaea maderae: Protein a immunoassay

Fat body maintained in vitro from adult female Leucophaea maderae synthesizes and secretes at least two peptides (275 and 220 kDa by SDS-PAGE) that can be photoaffinity labelled with the juvenile hormone analog 10-[10,11- 3H]epoxyfarnesyl diazoacetate ([ 3H]EFDA). To determine if the synthesis of these peptides by the fat body is a stage-dependent phenomenon, a protein A immunoassay (PAA assay) for quantifying rates of [ 3H]leucine incorporation into these peptides was set up and its specificity of binding monitored. To produce antibodies specific for these peptides, hybridoma technology was utilized. Hemolymph juvenile hormone-binding protein (JHBP) was semi-purified on sucrose gradients and used to immunize BALB/c mice. After the mice displayed a relatively high immunotiter to a JHBP solution, their spleens were used for the production of IgG-secreting hybridomas. Successful fusions were initially screened by using an enzyme-linked immunosorbent assay [ELISA], while the specificity of the secreted antibody was determined by using migration patterns of IgG-linked JHBPs on sucrose gradients and reactivity on Western blots. Fixed staphylococci were used as a solid phase adsorbent for isolating [ 3H]leucine-labelled antigen-antibody complexes. Identification of the isolated IgG-complexed [ 3H]leucine-peptides by SDS-PAGE fluorography demonstrated the specificity of the staphylococcal protein A immunoassay. Using this immunoassay we have measured rates of synthesis and secretion of [ 3H]leucine-JHBPs from fat body maintained in vitro and have found their synthesis to be stage-dependent. Fat bodies from early virgin females were least active, while fat bodies from mated females were most active. Injections of JH III into decapitated virgin females stimulated the synthesis of the JHBPs, while mineral oil injections had no effect.

Influence of castration and androgen treatment on the synthesis of prostatic binding protein and the concentration of its mRNA in the rat ventral prostate

The regulation by androgens of the synthesis of prostatic binding protein (PBP), an abundant secretory protein of the rat ventral prostate, was studied by measuring the rate of incorporation of [ 3H]leucine into total protein and into PBP by prostatie tissue in vitro. PBP-specific synthesis dropped to 30% of the intact level after 3 days and to 5.5% after 7 days. Administration of androgens to 7 day-castrated rats reversed these changes: 36% of the control value was reached after 3 days of treatment and 70% after 5 days. The mRNAs of PBP were measured under similar conditions by means of dot hybridization, using C 1-, C 2- and C 3-specific cDNA-clones. The 3 mRNAs followed a parallel course. They decreased to about 18% on the third day after castration and to about 2% after 7 days. Androgen treatment of 7-day castrated rats produced a small increase of these levels after 1 day. After 3 days 15% of the intact level was reached. The general similarity of the time course of PBP synthesis and PBP mRNA levels in castrated and androgen treated rats supports a transcriptional regulation mechanism, although there also is some evidence for an influence on protein synthesis in general.

Regulation of peptide transport in Escherichia coli: induction of the trp-linked operon encoding the oligopeptide permease.

Growth of Escherichia coli in medium containing leucine results in increased entry of exogenously supplied tripeptides into the bacterial cell. This leucine-mediated elevation of peptide transport required expression of the trp-linked opp operon and was accompanied by increased sensitivity to toxic tripeptides, by an enhanced capacity to utilize nutritional peptides, and by an increase in both the velocity and apparent steady-state level of L-[U-14C]alanyl-L-alanyl-L-alanine accumulation for E. coli grown in leucine-containing medium relative to these parameters of peptide transport measured with bacteria grown in media lacking leucine. Direct measurement of opp operon expression by pulse-labeling experiments demonstrated that growth of E. coli in the presence of leucine resulted in increased synthesis of the oppA-encoded periplasmic binding protein.

Phosphoinositides in mitogenesis: Neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation

Thrombin stimulates 32Pi incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bis-phosphate (PIP 2), and phosphatidylinositol (PI), and initiates DNA synthesis in hamster (NIL) fibroblasts at a half-maximal concentration of 125 ng/ml. Neomycin, which binds PIP 2 and PIP, inhibits both thrombin-stimulated initiation of cell proliferation and 32P incorporation into PI at concentrations above 2 mM without affecting thrombin binding, thymidine uptake, or cellular protein synthesis. At lower concentrations, neomycin inhibits thrombin-stimulated release of inositol 1,4,5-trisphosphate (IP 3), by selectively binding PIP 2, but does not inhibit 32P incorporation into PI or initiation of DNA synthesis. Phosphoinositide recycling and diacylglycerol release therefore appear necessary for initiation of cell proliferation by thrombin. IP 3-stimulated Ca ++ mobilization may not be required for thrombin mitogenesis, however, since neomycin can block IP 3 release without inhibiting initiation.

Inhibition of poly(A)-binding protein synthesis in Friend erythroleukemia cells subsequent to heat shock

When Friend erythroleukemia cells (FEC) are incubated at 43°C there is a rapid and nearly complete inhibition of protein synthesis which can be reversed when cells are returned to their normal growing temperature of 37°C. Examination of the recovery of FEC from heat shock indicates that most cellular mRNAs behave as a cohort and return to translation at approximately the same rate. We found a notable exception to this rule in the case of a 78 kDa basic protein (named protein A) whose rate of return to a normal synthetic rate is markedly inhibited subsequent to heat shock. We show that protein A corresponds to the 78 kDa polypeptide commonly found to be associated with the poly(A) tails of mammalian mRNA.

Calcium messenger system: an integrated view.

Calcium messenger system: an integrated view.

A mutation in the adenovirus type 5 DNA binding protein that fails to autoregulate the production of the dna binding protein

H5ts125 is a temperature-sensitive mutant of type 5 adenovirus whose defect maps in the structural gene of the viral DNA binding protein. Among a large number of temperature-independent revertants of this mutant, that plaque efficiently at 39° in HeLa cells, one revertant, r(ts125)13, has been studied in more detail. The synthesis and stability of the adenovirus DNA binding protein in HeLa cells infected with Ad5wt, H5ts125, and r(ts125)13 was investigated by pulse-chase experiments with [ 35S]methionine. In H5ts125-infected cells at the nonpermissive temperature about 1 1 2 to 2 1 2 - fold more DNA binding protein was synthesized in the pulse-labeling period than was detected in wild-type infected cells. Unlike the wild-type protein, the H5ts125 DNA binding protein was unstable and it was degraded during the chase period. In r13-infected HeLa cells, the DNA binding protein was overproduced 3- to 5-fold during the pulse-labeling time. The r13 DNA binding protein was stable and accumulated throughout the chase period. The r13 DNA binding protein was not overproduced and remained stable during a chase period, in human 293 cells on monkey CV-1 cells indicating that the faulty autoregulation of the r13 DNA binding protein synthesis was host cell or host range specific. These experiments provide genetic evidence for the adenovirus DNA binding proteins' ability to autoregulate its own synthesis. In addition the results suggest that cellular functions, interacting with the DNA binding protein, play a role in this autoregulatory event.

Transient enhanced synthesis of a cellular protein following infection of Aedes albopictus cells with Sindbis virus

Sindbis or Chikungunya virus infection of Aedes albopictus cells led to the synthesis of maximum amounts of intracellular virus structural protein at approximately 24 hr postinfection. Subsequently, viral protein synthesis declined and by 3 days postinfection little capsid protein was synthesized in infected cells. Starting at 2 days postinfection and for a period of 2–3 days, infected cells synthesized large amounts of a predominantly nuclear protein with molecular weight 43K (P43). In addition, smaller quantities of four other nuclear proteins with molecular weights 80K, 71K, 41K, and 26K were also transiently synthesized. Several lines of evidence showed that P43 is very closely related to, if not identical to a 43K protein found in the nuclei of uninfected cells. First, the 43K protein from uninfected cells and P43 were indistinguishable on the basis of their electrophoertic mobilities in both one- and two-dimensional separations in denaturing poly-acrylamide gels. Second, both P43 and the 43K protein generated the same digestion products after treatment with Staphylococcus aureus V8 protease. Heterogeneous nuclear ribonucleoprotein (hnRNP) particles were isolated from the nuclei of both uninfected and virus infected A. albopictus cells 3 days postinfection. HnRNP particles sedimented at 39 S and contained, as their major constituent, a protein which comigrated in both one- and two-dimensional gels with P43. These data indicate that alphavirus infection of A. albopictus cells leads to a transient enhancement in the synthesis of a 43K hnRNA binding protein.

Prostatic binding protein, polyamine, and DNA synthesis in rat ventral prostate cells.

Prostatic binding protein (PBP), polyamine, and DNA synthesis were examined in primary cultures of rat ventral prostate cells. Soon after aggregates of prostate cells were placed in culture, PBP synthesis fell dramatically and DNA synthetic activity in the epithelial cells increased. The amount of polyamines that were labeled with [3H] from [3H] ornithine fell transiently, but rose again at or before the peak of DNA synthesis. Individual 3H-labeled polyamines in cells and medium were dansylated and separated by thin-layer chromatography. The ratio of [3H] Spermidine plus [3H] Spermine to [3H] Putrescine in the culture medium declined as DNA synthesis increased. Ornithine decarboxylase (ODC) activity fell dramatically along with PBP synthesis even as DNA synthesis and 3H-labeled polyamines increased in the prostate-cell cultures. These results support others that suggest that high ODC activity in prostate epithelial cells is a correlate of prostate epithelial cell function rather than proliferation. However, prostate epithelial cells retain the capacity to synthesize significant levels of polyamines from ornithine (especially Putrescine) during proliferation even when ODC activity is low.

Effects of zinc in the synthesis of cadmium binding protein in cadmium-treated female rats: the relationship between cadmium binding protein concentration and cadmium-induced liver damage.

Sixty female rats were divided into 4 groups. The diet for group I was supplemented with 200 pg Cd per g diet and that for group II was supple-mented with 200 pg Zn per g diet and to the diet for group III were added 200 pg Cd and 200 pg Zn per g diet. Group IV was the control group fed normal diet. They were sacrificed at 11th, 26th and 40th week of exposure. Cd content in the liver of the rats exposed to Cd alone (group I) was higher than that of the rats exposed to Cd-F Zn (group III) at 26th and 40th week of exposure. Metallo-thionein (MT) concentration in the rats of group I was consistently higher than that of group III. At 40th week of exposure, MT fraction in the sephadex G-75 elution profile of group I showed a definite shoulder and this shoulder was not observed in the rats of group III. Pathological changes were observed in the liver of group I but not in group III. Present results suggest that the formation of additional molecular species of Cd containing protein is associated with Cd-induced hepatic damage.

Studies on the Toxicity and Binding Kinetics of Abrin in Normal and Epstein-Barr Virus-Transformed Lymphocyte Culture-I

The effects of treatment with varying doses of abrin, a Z > -galactose binding lectin, on DNA and protein synthesis of normal and Epstein-Barr virus (EBV)-transformed lymphocytes have been previously investigated. Using data on EBV-transformed lymphocyte cell density as a function of both time and dose of abrin, the authors introduced the concept of self- and cross-coupling metabolic variables as a means of understanding how abrin affected DNA and protein uptake. In this paper, the self-coupling constant is studied in more detail and the relationship between DNA and protein synthesis is further expanded. We find that there is a significant linear relationship between DNA and protein synthesis in normal lymphocyte culture as measured by abrin interaction in the culture. We further find that there is a much stronger relationship between these variables in EBV-transformed lymphocyte culture. This relationship is further examined, and possible analytic equations are expressed.

Status of the Pituitary-Adrenocortical-Liver Axis following Partial Hepatectomy

AbstractSince the liver is the primary site of steroid degradation and plasma binding protein synthesis, studies were undertaken to evaluate adrenocortical activity in partially hepatectomized (H) male rats. Two days after either 10, 30, 40, or 70% H, significant decreases in liver mass were observed, while elevations in total and free plasma corticosterone levels, adrenal corticosterone content, and adrenal weights were noted only in the 70% H group. Hepatic δ4-steroid hydrogenase activity was significantly depressed in both whole and microsomal, but not in the supernatant, fractions of the regenerating liver of the 70% H group. Although plasma ACTH levels were unchanged in these animals, the in vitro adrenocortical responsiveness to ACTH was increased. These data suggest that a portion of the increase in total plasma corticosterone levels post-70% H was due to a reduction in the hepatic capacity to inactivate corticosterone, while the remainder may be due to an increase in adrenocortical secretion as a ...

Studies on the toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 3.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of sites bound/EBV-lymphocyte needed to exert a biological influence upon the cell DNA synthesis lies between 59,264 and 370,000 sites/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 and 360,000 sites/cells.

Studies on the toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 1.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Activation, stimulation and relative toxicity factor indices are studied as well as possible relationships between the DNA and protein synthesis rates, as measured by simultaneous 3H-TdR and 14C-leucine uptake.

On the dynamics of abrin binding to receptor sites in normal and Epstein Barr Virus transformed lymphocyte cell cultures.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA an protein synthesis of normal and Epstein Barr Virus transformed lymphocytes has been investigated. Activation, stimulation, and relative toxicity factor indices are studied, as well as possible relationships between DNA and protein synthesis rates, as measured by simultaneous tritiated thymidine (3H-TdR) and 14C-leucine uptake. Studies of the two new indices, the metabolic self and cross coupling indices lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin receptor site concentrations. This prediction is supported by SEM morphological differences. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of receptors bound-EBV-lymphocyte needed to exert a biological influence lies in the interval 59,264 receptors/cell to 370.040 receptors/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 receptors/cell and 360,000 receptors/cell.

Studies on toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 2.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Studies of activation and stimulation indices, as well as two new indices; the metabolic self and cross-coupling indices, lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin binding site numbers. This conclusion is supported by SEM morphological differences.