Introductory Lecture: In Vitro Translation Analysis of Integral Membrane Proteins

Abstract

A method of in vitro translation scanning was applied to a variety of polytopic integral membrane proteins, a transition metal P type ATPase from Helicobacter pylori, the SERCA 2 ATPase, the gastric H⁺,K⁺ ATPase, the CCK-A receptor and the human ileal bile acid transporter. This method used vectors containing the N terminal region of the gastric H⁺,K⁺ ATPase or the N terminal region of the CCK-A receptor, coupled via a linker region to the last 177 amino acids of the β-subunit of the gastric H⁺,K⁺ ATPase. The latter contains 5 potential N-linked glycosylation sites. Translation of vectors containing the cDNA encoding one, two or more putative transmembrane domains in the absence or presence of microsomes allowed determination of signal anchor or stop transfer properties of the putative transmembrane domains by the molecular weight shift on SDS PAGE. The P type ATPase from Helicobacter pylori showed the presence of 8 transmembrane segments with this method. The SERCA 2 Ca²⁺ ATPase with this method had 9 transmembrane co-translational insertion domains and coupled with other evidence these data resulted in a 11 transmembrane segment model. Translation of segments of the gastric H⁺,K⁺ ATPase provided evidence for only 7 transmembrane segments but coupled with other data established a 10 membrane segment model. The G7 protein, the CCK-A receptor showed the presence of 6 of the 7 transmembrane segments postulated for this protein. Translation of segments of the human ileal bile acid transporter showed the presence of 8 membrane insertion domains. However, translation of the intact protein provided evidence for an odd number of transmembrane segments, resulting in a tentative model containing 7 or 9 transmembrane segments. Neither G7 type protein appeared to have an arrangement of sequential topogenic signals consistent with the final assembled protein. This method provides a useful addition to methods of determining membrane domains of integral membrane proteins but must in general be utilized with other methods to establish the number of transmembrane α-helices.     

Radiation Damage to the Erythrocyte Membrane: The Effects of Medium and Cell Concentrations

Human erythrocytes suspended in plasma, or in phosphate buffered saline (PBS), were exposed to ionizing radiation. Potassium leakage from irradiated erythrocytes is significantly higher in PBS than in plasma. The potassium leakage decreases when PBS is gradually replaced by plasma. These findings suggest that some of the plasma constituents have radioprotective properties. The potassium leakage per cell is independent of the hematocrit, Hct. The potassium leakage is attributed to the formation of radiation defects in the membrane. Analysis of the effect of radiation dose, plasma and cell concentrations on the product of the number and surface area of the radiation defects indicates that the radiation damage is mainly due to the direct formation of free radicals in the cell membrane. The radioprotective effect of plasma is attributed to surface reactions of these free radicals with plasma constituents adsorbed on the membrane.     

Radiation Damage to the Erythrocyte Membrane: The Effects of Medium and Cell Concentrations

Human erythrocytes suspended in plasma, or in phosphate buffered saline (PBS), were exposed to ionizing radiation. Potassium leakage from irradiated erythrocytes is significantly higher in PBS than in plasma. The potassium leakage decreases when PBS is gradually replaced by plasma. These findings suggest that some of the plasma constituents have radioprotective properties. The potassium leakage per cell is independent of the hematocrit, Hct. The potassium leakage is attributed to the formation of radiation defects in the membrane. Analysis of the effect of radiation dose, plasma and cell concentrations on the product of the number and surface area of the radiation defects indicates that the radiation damage is mainly due to the direct formation of free radicals in the cell membrane. The radioprotective effect of plasma is attributed to surface reactions of these free radicals with plasma constituents adsorbed on the membrane.     

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

Abstract: The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TER) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one-to-one mixtures of these proteins. Moreover, a one-to-one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (∼350 Ω× cm²) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one-to-one combinations of these proteins elevated transcellular resistances significantly (2.3- to 2.9-fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (∼1,000 Ω× cm²) could, by contrast, be increased only 1.1- to 1.2-fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood-brain barrier.     

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

Studies of in vitro processing of precursors of the major chlorophyll a/b-binding polypeptides of Chlamydomonas reinhardtii y-1 were undertaken to define the precursor-product relationships. Analysis of translates, prepared from C. reinhardtii poly(A)-rich RNA in a rabbit reticulocyte lysate system, which were incubated with the soluble fraction from C. reinhardtii cells, showed that the 31,500 relative molecular mass (M_r) precursor was converted to the M_r 29,500 thylakoid membrane polypeptide whereas the M_r 30,000 precursor was converted to the M_r 26,000 product. Furthermore, the M_r 31,500 polypeptide, when bound to antibodies, was not processed to the mature polypeptide of M_r 29,500, although the presence of antibodies did not prevent the precursor of M_r 30,000 from being converted to the mature M_r 26,000 polypeptide. The mature fraction of M_r 26,000, was separated into two bands corresponding to polypeptides 16 and 17 in the electrophoretic system of Chua and Bennoun (1975 Proc Natl Acad Sci USA 72: 2175-2179).

Processing activity was present in the soluble fraction obtained from cells grown in the light or in the dark. Therefore, processing of the precursor polypeptides does not appear to be involved in the regulation by light of the accumulation of these polypeptides in thylakoid membranes.

     

The Negative Effect of Soy Extract on Erythrocyte Membrane Fluidity: An Electron Paramagnetic Resonance Study

A decrease of erythrocyte membrane fluidity can contribute to the pathophysiology of hypertension. Soy products, which are used as alternative therapeutics in some cardiovascular conditions, contain various isoflavones (genistein, daidzein, and their glucosides, genistin and daidzin), which can incorporate cellular membrane and change its fluidity. The aim of this study was to examine the effects of soy extract (which generally corresponds to the soy products of isoflavone composition) on erythrocyte membrane fluidity at graded depths. We used electron paramagnetic resonance spectroscopy and fatty acid spin probes (5-DS and 12-DS), the spectra of which are dependent on membrane fluidity. After being treated with soy extract, erythrocytes showed a significant (P = 0.016) decrease of membrane fluidity near the hydrophilic surface, while there were no significant changes of fluidity in deeper hydrophobic membrane regions. These results suggest that soy products containing high levels of genistein and isoflavone glucosides may not be suitable for use in hypertension because they decrease erythrocyte membrane fluidity.     

红细胞膜肌醇磷酯结合蛋白的研究

本文用~8H-肌醇标记红细胞膜,分别用薄层层析及SDS-PAGE对肌醇磷酯在红细胞膜上的分布作了探讨。发现一种在脂双层内,另一种是与膜蛋白结合(即肌醇磷酯结合蛋白)作为膜蛋白的锚(anchor)而存在,并用抗AchE及DAF单克隆抗体作免疫印渍实验,在电泳中~3H-肌醇磷酯所在部位与AchE及DAF相符。这结果与文献中报道AchE及DAF为肌醇磷酯结合蛋白一致。至于这两种形式不同的肌醇磷酯的相互关系还有待研究。     

Highly Purified pp60src Induces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro

The src gene product of Rous sarcoma virus (pp60(src)) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.     

In Vitro Induced Anaerobic Resistance to Metronidazole In Trichomonas Vaginalis

ABSTRACT. Resitance to metronidazole detectable under anaerobic conditions was induced in two Trichomonas vaginalis strains (TV 10-02 and MRP-2) by cultivation at gradually increasing pressure of the drug (1-100 μ/ml) for 12 to 21 months. the resistant derivatives reproduced in anaerobic trypticase-yeast-extract-maltose medium at 100 μ/ml metronidazole and showed very high values of minimal lethal concentration for metronidazole in anaerobic in vitro assays (556-1,600 μ/ml at 48-h exposure to the drug). Stepwise selection was necessary to develop the resistance in either strain. Attempts to induce resistance by prolonged maintenance of trichomonads with constant, low or moderate drug concentrations (3-10 μ/ml) were unsuccessful. Freshly developed resistance to high concentrations of metronidazole was unstable in absence of drug pressure as well as after cryopreservation. Development of stable resistance required further cultivation at 100 μ/ml metronidazole. Unstable substrains did not revert to original susceptibility. They retained a moderate level of resistance, being able to grow at 10 μ/ml metronidazole. the strains with fully developed resistance had no activity of the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase and ceased uptake of [¹⁴C]-metronidazole. These findings indicate that the pyruvate oxidizing pathway responsible for metronidazole activation was inactivated and metabolism of the drug stopped.     

In Vitro Synthesis of Adenovirus Core Proteins

mRNA extracted from polysomes of KB cells at late stages of productive infection with adenovirus type 2 was translated in a cell-free system derived from Krebs II ascites cells. Two of the polypeptides obtained in this reaction corresponded to the adenovirus core protein V and the precursor to core protein VII. The following criteria were used to establish identity between the in vitro products and the virion proteins: comigration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis, cochromatography in sodium dodecyl sulfate-hydroxyapatite, specific immunoprecipitation of the precursor to core protein VII, and tryptic peptide analysis.     

Effects of Calcium, Strontium, and Barium Ions on Phosphorylation of Hippocampal Proteins In Vitro

Abstract: Calcium ion alone or in the presence of added calmodulin stimulated in vitro transfer of ³²P from [γ³²P]ATP into several proteins of mitochondrial and synaptosomal particulate fractions from rat brain. Strontium ion was capable of substituting for calcium ion in this stimulation, but barium ion lacked this capacity. These results bring into question the hypothesis that calciumdependent protein phosphorylation of synaptic proteins is intrinsic to neurotransmitter release during neurotransmission, but they do not rule out that possibility.     

Ionising Radiation Immediately Impairs Synaptic Plasticity-Associated Cytoskeletal Signalling Pathways in HT22 Cells and in Mouse Brain: An In Vitro/In Vivo Comparison Study

Patients suffering from brain malignancies are treated with high-dose ionising radiation. However, this may lead to severe learning and memory impairment. Preventive treatments to minimise these side effects have not been possible due to the lack of knowledge of the involved signalling pathways and molecular targets. Mouse hippocampal neuronal HT22 cells were irradiated with acute gamma doses of 0.5 Gy, 1.0 Gy and 4.0 Gy. Changes in the cellular proteome were investigated by isotope-coded protein label technology and tandem mass spectrometry after 4 and 24 hours. To compare the findings with the in vivo response, male NMRI mice were irradiated on postnatal day 10 with a gamma dose of 1.0 Gy, followed by evaluation of the cellular proteome of hippocampus and cortex 24 hours post-irradiation. Analysis of the in vitro proteome showed that signalling pathways related to synaptic actin-remodelling were significantly affected at 1.0 Gy and 4.0 Gy but not at 0.5 Gy after 4 and 24 hours. We observed radiation-induced reduction of the miR-132 and Rac1 levels; miR-132 is known to regulate Rac1 activity by blocking the GTPase-activating protein p250GAP. In the irradiated hippocampus and cortex we observed alterations in the signalling pathways similar to those in vitro. The decreased expression of miR-132 and Rac1 was associated with an increase in hippocampal cofilin and phospho-cofilin. The Rac1-Cofilin pathway is involved in the modulation of synaptic actin filament formation that is necessary for correct spine and synapse morphology to enable processes of learning and memory. We suggest that acute radiation exposure leads to rapid dendritic spine and synapse morphology alterations via aberrant cytoskeletal signalling and processing and that this is associated with the immediate neurocognitive side effects observed in patients treated with ionising radiation.     

Entropic Imaging of Cataract Lens: An In Vitro Study

Phacoemulsification is a common surgical method for treating advanced cataracts. Determining the optimal phacoemulsification energy depends on the hardness of the lens involved. Previous studies have shown that it is possible to evaluate lens hardness via ultrasound parametric imaging based on statistical models that require data to follow a specific distribution. To make the method more system-adaptive, nonmodel-based imaging approach may be necessary in the visualization of lens hardness. This study investigated the feasibility of applying an information theory derived parameter – Shannon entropy from ultrasound backscatter to quantify lens hardness. To determine the physical significance of entropy, we performed computer simulations to investigate the relationship between the signal-to-noise ratio (SNR) based on the Rayleigh distribution and Shannon entropy. Young''s modulus was measured in porcine lenses, in which cataracts had been artificially induced by the immersion in formalin solution in vitro. A 35-MHz ultrasound transducer was used to scan the cataract lenses for entropy imaging. The results showed that the entropy is 4.8 when the backscatter data form a Rayleigh distribution corresponding to an SNR of 1.91. The Young''s modulus of the lens increased from approximately 8 to 100 kPa when we increased the immersion time from 40 to 160 min (correlation coefficient r = 0.99). Furthermore, the results indicated that entropy imaging seemed to facilitate visualizing different degrees of lens hardening. The mean entropy value increased from 2.7 to 4.0 as the Young''s modulus increased from 8 to 100 kPa (r = 0.85), suggesting that entropy imaging may have greater potential than that of conventional statistical parametric imaging in determining the optimal energy to apply during phacoemulsification.     

Cleavage of Viral Precursor Proteins In Vivo and In Vitro

The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages.     

Molecular Interaction of S-100 Proteins with Microtubule Proteins In Vitro

Several procedures were employed to examine the in vitro interaction between S-100 proteins and microtubule proteins. Binding of S-100 to tau factors was observed under all experimental conditions. S-100 binding to microtubule-associated protein 2 (MAP2) was best detected by exposing nitrocellulose-immobilized MAP2 or MAPs to either 125I-labeled S-100 or biotinylated S-100. S-100 binding to tubulin was detected when the two protein fractions were first incubated with each other followed by exposure to the bifunctional cross-linker disuccinimidylsuberate, and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfered onto nitrocellulose paper. By this procedure, complex formation between S-100 and tubulin, as well as between S-100 and a relatively low-molecular-weight MAP, was evidenced by immunoblotting using an anti-S-100 antiserum. Alternatively, complex formation between biotinylated S-100 and either tubulin or MAPs was visualized by means of avidin-peroxidase, after SDS-PAGE of the complex mixtures and transfer of the separated proteins onto nitrocellulose. The interaction between S-100 and tubulin was strictly Ca2+ dependent, and resistant to high concentrations of KCl, colchicine, or vinblastine.     

An In Vitro Test for Temperature Sensitivity and Resistance to Meloidogyne incognita in Tomato

An in vitro root explant tissue culture technique is described for determining susceptibility of tomato (Lycopersicon esculentum Mill.) breeding lines and cultivars to the root-knot nematode Meloidogyne incognita. Root explants were taken from 2-day-old seedlings cultured for 30 days at 28 C on Gamborg''s B-5 medium with or without nematode inoculum. The remaining portion of the root and stem from the excised root explants was transferred to soil in pots and grown to maturity in the greenhouse. In vitro root explants were evaluated for growth and occurrence of juveniles, adults, and egg masses. The regenerated plants were used to produce more seed, The proposed technique is simple, reliable, and adapted to routine screening of large numbers of F₁ and F₂ samples, and it utilizes less space than tests performed on intact plants in the greenhouse or growth chamber. Evidence is presented also on the breakdown of resistance to M. incognita under high temperature stress using this in vitro root explant technique.     

An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.