Course Of Incorporation Research Articles

A prospective, randomized, double blind, placebo-controlled evaluation of the effects of an n-3 essential fatty acids supplement (Agepi® ω3) on clinical signs, and fatty acid concentrations in the erythrocyte membrane, hair shafts and skin surface of dogs with poor quality coats

Canine haircoat quality, the time course of incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into the canine erythrocyte membrane, changes in total lipids content on hair shafts and of neutral lipids on the skin surface, were investigated after administrating an n-3 fatty acids supplement. Twenty-four dogs with poor haircoat received a placebo or n-3 oil capsules (110 mg EPA/68 mg DHA) daily for 90 days. Clinical assessments and tissue sampling were performed monthly from day (D)0 to D180. The clinical score in treated dogs was significantly reduced from D60, then attained a plateau and returned to baseline levels on D180. EPA and DHA contents in the erythrocyte membrane increased significantly from D30 and decreased rapidly after supplement withdrawal in treated dogs. Total lipids on the hair shaft increased progressively in the supplemented group. EPA/DHA supplements impact blood and hair fatty acids important for haircoat quality in dogs.

Daydreams incorporate recent waking life concerns but do not show delayed ('dream-lag') incorporations.

This study investigates the time course of incorporation of waking life experiences into daydreams. Thirty-one participants kept a diary for 10 days, reporting major daily activities (MDAs), personally significant events (PSEs) and major concerns (MCs). They were then cued for daydream, Rapid Eye Movement (REM) and N2 dream reports in the sleep laboratory. There was a higher incorporation into daydreams of MCs from the previous two days (day-residue effect), but no day-residue effect for MDAs or PSEs, supporting a function for daydreams of processing current concerns. A day-residue effect for PSEs and the delayed incorporation of PSEs from 5 to 7 days before the dream (the dream-lag effect) have previously been found for REM dreams. Delayed incorporation was not found in this study for daydreams. Daydreams might thus differ in function from REM sleep dreams. However, the REM dream-lag effect was not replicated here, possibly due to design differences from previous studies.

Spirilloxanthin incorporation into the LH2 and LH1-RC pigment-protein complexes from a purple sulfur bacterium Allochromatium minutissimum

Incorporation of spirilloxanthin into carotenoidless LH2 and LH1-RC complexes from a purple sulfur bacterium Allochromatium (Alc.) minutissimum was studied. Carotenoidless cells of Alc. minutissimum were obtained using diphenylamine, a carotenoid biosynthesis inhibitor. In the course of incorporation of the carotenoid mixture, the composition of which corresponded to that of Alc. minutissimum control photosynthetic membranes, no selective incorporation of spirilloxanthin into the LH1-RC complex was detected. It is assumed that in vivo carotenoids are not incorporated into the LH2 and LH1-RC complexes from a common pool. Pure spirilloxanthin destroys both the LH2 and LH1-RC complexes. Within the concentration range of spirilloxanthin in the incorporated mixture from 27% to 52%, it was found to be incorporated into the LH2 and LH1-RC complexes with the efficiency of 13% and 33%, respectively. The possible existence of different sites of assembly for the LH2 and LH1-RC complexes is discussed, as well as of two fractions of LH2 complexes, in one of which rhodopin may be integrated, and in the other (minor) one, spirilloxanthin.

СТРАТЕГІЧНЕ ЗНАЧЕННЯ УКРАЇНСЬКИХ ЕТНІЧНИХ ТЕРИТОРІЙ У ПРОЦЕСІ КАНСТЕЛЯЦІЇ ЦЕНТРАЛЬНОЇ ЄВРОПИ В 1920-ТІ РР.

In this article the problem of Ukrainian impact in the process of the formation of security system in Central Europe in the 1920s has been studied. The strategic motifs in the course of incorporation of Ukrainian ethnic territories to the territories of Poland, Romania, and Czechoslovakia during the process of formation of Versailles system have been researched. Particular attention has also been paid to the problem of formation of allied relations between the counties of Central Europe, the main target of which was opposition to the German and Hungarian revanchism and Soviet expansionistic efforts. The role of the regional alliances in the Central Europe, France, Great Britain, Germany and USSR has been analyzed.

Stable isotope labeling by amino acids in cell culture-based liquid chromatography–mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures

Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography–mass spectrometry (LC–MS) method to measure the fraction of [13C6]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [13C6]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.

Abstract 2222: Analysis of the autophosphorylation-induced calcium-independent activity of human elongation factor 2 kinase (eEF-2K) - a therapeutic target for breast cancer.

Abstract Breast cancer is the most common malignancy in women in the Western world, with over 40,000 deaths attributed to this disease. Studies have demonstrated that breast cancer mitogens regulate the activity of eukaryotic elongation factor 2 kinase (eEF-2K). The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. We have recently shown for the first time that the targeting of eEF-2K in an in vivo orthotopic model inhibits growth of established breast cancer tumors and sensitizes the tumors to doxorubicin, an important agent in a number of chemotherapy regimens. Our data suggest that eEF-2K may enhance tumorigenesis through the up-regulation of pro-tumorigenic proteins and pathways including cyclin D1, c-Myc, c-Src/FAK and Akt, whose dysregulation are associated with a poor prognosis in breast cancer. Thus, the targeting of eEF-2K in breast cancer cells has potential as a therapeutic approach for the treatment of breast cancer. In order to develop inhibitors of eEF-2K, an understanding of the regulation of enzyme activity is essential. eEF-2K is an atypical calcium/calmodulin-dependent protein kinase (CaMK-III), that through its phosphorylation of elongation factor 2 (eEF-2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The purpose of our study was to analyze the role of calcium/calmodulin-stimulated autophosphorylation in the generation of a calcium-independent form of eEF-2K. Analysis of the kinase activity using [γ-32P]ATP indicates that autophosphorylation of eEF-2K induces calcium-independent activity. Site-directed mutagenesis of the autophosphorylation sites to alanine and aspartate suggests that the site responsible for this calcium-independent activity is Ser-500. Moreover, using a phospho-specific antibody we have determined that the time course of incorporation of phosphate at Ser-500 correlates with the induction of calcium-independent activity. Surprisingly, calmodulin appears to be essential for this calcium-independent activity of eEF-2K. Mechanistic studies indicate that phosphorylation of eEF-2K on Ser-500 increases its affinity for calmodulin in the absence of calcium, potentially by decreasing its rate of dissociation. We have also found that eEF-2K is indeed phosphorylated on Ser-500 in cells, and we are currently studying the dynamic nature of phosphorylation at this site, and its effect on stability of the enzyme and its affinity for calmodulin. Citation Format: Clint D. J. Tavares, Scarlett B. Ferguson, Rebecca M. Wellmann, Tamer S. Kaoud, John P. O'Brien, Bulent Ozpolat, Jennifer S. Brodbelt, Kevin N. Dalby. Analysis of the autophosphorylation-induced calcium-independent activity of human elongation factor 2 kinase (eEF-2K) - a therapeutic target for breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2222. doi:10.1158/1538-7445.AM2013-2222

Isotopomer analysis of lipid biosynthesis by high resolution mass spectrometry and NMR

We have coupled 2D-NMR and infusion FT-ICR-MS with computer-assisted assignment to profile 13C-isotopologues of glycerophospholipids (GPL) directly in crude cell extracts, resulting in very high information throughput of >3000 isobaric molecules in a few minutes. A mass accuracy of better than 1 ppm combined with a resolution of 100,000 at the measured m/ z was required to distinguish isotopomers from other GPL structures. Isotopologue analysis of GPLs extracted from LCC2 breast cancer cells grown on [U- 13C]-glucose provided a rich trove of information about the biosynthesis and turnover of the GPLs. The isotopologue intensity ratios from the FT-ICR-MS were accurate to ≈1% or better based on natural abundance background, and depended on the signal-to-nose ratio. The time course of incorporation of 13C from [U- 13C]-glucose into a particular phosphatidylcholine was analyzed in detail, to provide a quantitative measure of the sizes of glycerol, acetyl CoA and total GPL pools in growing LCC2 cells. Independent and complementary analysis of the positional 13C enrichment in the glycerol and fatty acyl chains obtained from high resolution 2D NMR was used to verify key aspects of the model. This technology enables simple and rapid sample preparation, has rapid analysis, and is generally applicable to unfractionated GPLs of almost any head group, and to mixtures of other classes of metabolites.

Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase

It has recently been demonstrated that the anti-herpetic drug acyclovir (ACV) also displays antiviral activity against the human immunodeficiency virus type 1 (HIV-1). The triphosphate form of ACV is accepted by HIV-1 reverse transcriptase (RT), and subsequent incorporation leads to classical chain termination. Like all approved nucleoside analogue RT inhibitors (NRTIs), the selective pressure of ACV is associated with the emergence of resistance. The V75I mutation in HIV-1 RT appears to be dominant in this regard. By itself, this mutation is usually not associated with resistance to currently approved NRTIs. Here we studied the underlying biochemical mechanism. We demonstrate that V75I is also selected under the selective pressure of a monophosphorylated prodrug that was designed to bypass the bottleneck in drug activation to the triphosphate form (ACV-TP). Pre-steady-state kinetics reveal that V75I discriminates against the inhibitor at the level of catalysis, whereas binding of the inhibitor remains largely unaffected. The incorporated ACV-monophosphate (ACV-MP) is vulnerable to excision in the presence of the pyrophosphate donor ATP. V75I compromises binding of the next nucleotide that can otherwise provide a certain degree of protection from excision. Collectively, the results of this study suggest that ACV is sensitive to two different resistance pathways, which warrants further investigation regarding the detailed resistance profile of ACV. Such studies will be crucial in assessing the potential clinical utility of ACV and its derivatives in combination with established NRTIs.

Description of nitrogen incorporation and release in ADM1

ADM1 represents a universally applicable biokinetic model for the mathematical description of anaerobic digestion of different types of organic substrates. Digestion of particulate composites is described as a five-stage process involving disintegration, hydrolysis, acidogenesis, acetogenesis and methanogenesis, of which the last three process steps are represented by growth kinetics of the specific degrading biomass. Decay of the produced biomass according to ADM1 is depicted by a recycle mass flux to the composite particulate substrate. Consequently two different actions are lumped into one process describing both conversion of feed substrate (depending primarily on influent characterisation) and generation of decay products (depending on digestion performance). In this presentation the introduction of a separate compound of inert decay products in analogy to ASM1 is suggested. Model calibration of separately monitored digestion of primary and secondary sludge (nitrogen content 0.030 g N/g TSS and 0.051 g N/g TSS, respectively) reveals the advantage of a clear distinction of disintegration and decay. The fate of nitrogen in the course of incorporation and release (0.016 g N/g TSS compared to 0.028 g N/g TSS) during digestion processes is comprehensible and the final ammonia concentration in the rejection water becomes predictable.

Metabolism of Myeloperoxidase-derived 2-Chlorohexadecanal

Numerous studies have suggested relationships between myeloperoxidase (MPO), inflammation, and atherosclerosis. MPO-derived reactive chlorinating species attack membrane plasmalogens releasing alpha-chloro fatty aldehydes including 2-chlorohexadecanal (2-ClHDA), which have been found to accumulate in activated neutrophils, activated monocytes, infarcted myocardium and human atheromas. The present study employed synthetically prepared 2-Cl-[3H]-HDA as well as stable isotope-labeled 2-ClHDA to elucidate the metabolism of 2-ClHDA. The results herein demonstrate that human coronary artery endothelial cells oxidize and reduce 2-ClHDA to its respective chlorinated fatty acid (alpha-ClFA) and chlorinated fatty alcohol (alpha-ClFOH). Within the first hour of incubations of human coronary artery endothelial cells with 2-Cl-[3H]-HDA, the label was incorporated into the alpha-ClFOH and alpha-ClFA pools. After 1 h, the radiolabel was predominantly found in the alpha-ClFOH pool. Cell-derived alpha-ClFOH and alpha-ClFA were also released into the cell culture medium. Additionally, chlorinated fatty acid was incorporated into complex endothelial cell glycerolipids, including monoglycerides, triglycerides, phosphatidylcholine, and phosphatidylethanolamine. The oxidation and reduction of 2-ClHDA to alpha-ClFA and alpha-ClFOH, respectively, was further supported by mass spectrometric analyses of human coronary artery endothelial cells incubated with either 2-ClHDA or stable isotope-labeled 2-ClHDA (2-Cl-[d4]-HDA). 2-ClHDA was also oxidized to alpha-ClFA and reduced to alpha-ClFOH in both control and phorbol 12-myristate 13-acetate-stimulated neutrophils. Taken together, these results show that a family of chlorinated lipidic metabolites is produced from alpha-chloro fatty aldehydes derived from reactive chlorinating species targeting of plasmalogens. These metabolites are incorporated into complex lipids and their biological roles may provide new insights into MPO-mediated disease.

Time course of incorporation of n-3 PUFA from linseed in pigs and effects on D9-desaturase activity and pork odours

The n-3 polyunsaturated fatty acids (PUFA) are healthy nutrients which can be increased in pork by feeding sources such as linseed to the growing animal. The levels achieved depend on many factors such as the concentrations of lipid classes in tissues (eg phospholipids containing high PUFA levels are more abundant in muscle than adipose tissue) competition for incorporation with n-6 PUFA and possible inhibitory effects of PUFA on synthesis of saturated and monounsaturated fatty acids. This study examined the time course of the incorporation of n-3 PUFA into tissue lipids and the effects on the major synthetic enzyme D9-desaturase. The effects on pork odour were also studied.

Unique Substrate Specificities of Two Adjacent Glutamine Residues in EAQQIVM for Transglutaminase: Identification and Characterization of the Reaction Products by Electrospray Ionization Tandem Mass Spectrometry

Reversed-phase HPLC (RP-HPLC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) were used to characterize the transglutaminase (TGase)-catalyzed dual modification of a peptide (EAQQIVM, named FibN) with monodansylcadaverine (MDC). The synthesized FibN peptide, which was derived from the N-terminal sequence of fibronectin, was used as the substrate for a guinea pig liver TGase (G-TGase). The time course of incorporation of MDC into FibN, detected by RP-HPLC, indicated two separate fluorescent product peaks. ESI-MS analysis of the isolated fractions indicated that products represented MDC-incorporated FibN molecules in molar ratios of 1:1 ((MDC)–FibN) and 2:1 ((MDC)2–FibN). A sequence analysis of MDC–FibN, using ESI-MS/MS, showed that the first modified residue in FibN was mainly Gln3. The kinetic analysis of MDC incorporation suggested that dual incorporation would occur by mainly one route. A one-dimensional 1H NMR comparison of MDC–FibN and unmodified FibN suggested that the first incorporation of MDC at Gln3 altered the substrate reactivity of the Gln4 residue in FibN for the G-TGase-catalyzed reaction. Thus, a detailed analysis of the peptide products using RP-HPLC and ESI-MS/MS should provide a powerful tool for exploring the mechanism of the substrate requirements of TGases.

Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus

Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [ 35S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells.

The time course of incorporation of [14C]arachidonic acid and [3H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [14C]arachidonic acid and [3H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.

The exogenous surfactant Curosurf enhances phosphatidylcholine content in isolated type II cells

The exogenous surfactant, Curosurf, contains proteins as well as phospholipids. We investigated the possibility that these might affect the reutilization of exogenous phospholipid by type II alveolar cells isolated from rat lung. The time course of incorporation into lamellar bodies of radioactivity from tritiated dipalmitoyl-phosphatidylcholine (DPPC) contained within liposomes was studied. Rates of uptake were compared between liposomes prepared from pure phospholipids and from Curosurf. The incorporation of labelled choline into newly synthesized phosphatidylcholine was also determined in the presence of both preparations. The rate of DPPC incorporation over the first 4 h was the same, but, after 6 and 8 h, the radioactivity associated with lamellar bodies was about 40% higher from Curosurf liposomes. By contrast, both Curosurf and the phospholipid mixture enhanced choline incorporation into phosphatidylcholine to the same extent. We conclude that Curosurf enhances the surfactant-related phosphatidylcholine content of type II cells by two mechanisms, one of which depends on the presence of proteins in exogenous surfactant. The difference in incorporation of radioactivity from liposome-associated labelled dipalmitoyl-phosphatidylcholine cannot be explained just by an increase in reutilization of choline from degraded dipalmitoyl-phosphatidylcholine.

Proteasome Inhibition Leads to a Heat-shock Response, Induction of Endoplasmic Reticulum Chaperones, and Thermotolerance

The accumulation of misfolded proteins in the cytosol leads to increased expression of heat-shock proteins, while accumulation of such proteins in the endoplasmic reticulum (ER) stimulates the expression of many ER resident proteins, most of which function as molecular chaperones. Recently, inhibitors of the proteasome have been identified that can block the rapid degradation of abnormal cytosolic and ER-associated proteins. We therefore tested whether these agents, by causing the accumulation of abnormal proteins, might stimulate the expression of cytosolic heat-shock proteins and/or ER molecular chaperones and thereby induce thermotolerance. Exposure of Madin-Darby canine kidney cells to various proteasome inhibitors, including the peptide aldehydes (MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal) and lactacystin, inhibited the degradation of short-lived proteins and increased markedly the levels of mRNAs encoding cytosolic heat-shock proteins (Hsp70, polyubiquitin) and ER chaperones (BiP, Grp94, ERp72), as shown by Northern blot analysis. However, inhibitors of cysteine proteases (E64), serine proteases (leupeptin), or metalloproteases (1, 10-phenanthroline) had no effect on the levels of these mRNAs. The relative efficacies of the peptide aldehyde inhibitors in inducing these mRNAs correlated with their potencies against the proteasome. Furthermore, reduction of the aldehyde group of MG132 decreased its inhibitory effect on proteolysis and largely prevented the induction of these mRNAs. Although treatment with the proteasome inhibitors caused rapid increases in mRNA levels (as early as 2 h after treatment with MG132), the inhibitors did not detectably affect total protein synthesis, total protein secretion, ER morphology, or the retention of ER-lumenal proteins, even after 18 h of treatment. Together, the findings suggest that inhibition of proteasome function induces heat-shock proteins and ER chaperones due to the accumulation of sufficient amounts of abnormal proteins and/or the inhibition of degradation of a key regulatory factor (e.g. heat-shock factor). Since expression of heat-shock proteins can protect cells from thermal injury, we tested whether the proteasome inhibitors might also confer thermotolerance. Treatment of cells with MG132 for as little as 2 h, markedly increased the survival of cells subjected to high temperatures (up to 46 degrees C). Thus, these agents may have applications in protecting against cell injury.

Accumulation of the xenobiotic anabolic steroids ethinyloestradiol and methyltestosterone in calf hair

An analytical method to detect the illegal application of anabolic steroids in cattle by hair analysis was developed. The time course of incorporation of the orally active xenobiotic steroids ethinyl‐oestradiol (EE2) and methyltestosterone (MT) into growing hair, the duration of their detection by hair analysis and the influence of hair pigmentation on steroid concentrations were investigated in detail. Female veal calves of different coat colour were fed with 3.5 μg of EE2 kg‐1 of body weight and 35 μg of MT kg‐1 of body weight, twice daily, for 10 days. Before, during and after treatment, hair samples were obtained and analyzed for EE2 and MT residues. An appropriate method was developed to extract EE2 and MT from hair. The extraction procedure consisted of liquid‐liquid and solid‐phase extraction and was followed by an essential high‐pressure liquid chromatography step for further purification of the extracts. The final quantification was carried out with specific enzyme immunoassays. EE2 and MT residues could be detected in hair from day 4 (approximately 1 ng of EE2 g‐1 of hair and 5 ng of MT g‐1) of the experiment up to day 38 (approximately 0.3 ng of EE2 g‐1) and day 94 (approximately 0.5 ng of MT g‐1) respectively. The plasma concentrations of MT during the treatment were about 10‐fold higher than the plasma concentrations of EE2. Steroid accumulation in hair seemed to depend on the plasma levels of the steroids applied because the accumulation of MT in hair was higher (approximately 7 ng g‐1) than the accumulation of EE2 (approximately 1 ng g‐1). In addition, the tracking of MT by hair analysis was possible for a much longer duration (up to day 94 of the experiment) than the detection of EE2 (day 38). There was no influence of pigmentation on the steroid concentrations in hair. These results suggest that hair analysis is a powerful means with which to detect and track the illegal use of anabolic steroids in animal production, horse‐racing and sports.

Kinetic Resolution of the Incorporation of the D1 Protein into Photosystem II and Localization of Assembly Intermediates in Thylakoid Membranes of Spinach Chloroplasts

The chloroplast-encoded D1 protein of photosystem II (PSII) has a much higher turnover rate than the other subunits of the PSII complex as a consequence of photodamage and subsequent repair of its reaction center. The replacement of the D1 protein in existing PSII complexes was followed in two in vitro translation systems consisting of isolated chloroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed translation elongation, release of proteins from the ribosomes, and subsequent incorporation of newly synthesized products into PSII (sub)complexes. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gradient centrifugation. Immediately after termination of translation, the D1 protein was found both unassembled in the membrane as well as already incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII reaction center components. Later steps in the reassembly of PSII were clearly post-translational and sequential. Different rate-limiting steps in the assembly process were found to be related to the depletion of nuclear encoded and stromal components as well as the lateral migration of subcomplexes within the heterogeneous thylakoid membrane. The slow processing of precursor D1 in the thylakoid translation system revealed that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precursor could take place. The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was clearly post-translational in both translation systems. Radiolabeled assembly intermediates smaller than the PSII core complex were found to be located in the stroma-exposed thylakoid membranes, the site of protein synthesis. Larger PSII assembly intermediates were almost exclusively located in the appressed regions of the membranes.

Rapid turnover of myosin light chain phosphate during cross-bridge cycling in smooth muscle

The rate of phosphatase-mediated dephosphorylation of the regulatory light chain of smooth muscle myosin was determined under nearly steady-state conditions in permeabilized muscles, from the time course of incorporation of 33P-labeled phosphate into the light chain after the photolytic release of [gamma-33P]ATP from high specific activity caged [gamma-33P]ATP. The extent of myosin light chain phosphorylation is unchanged, and, if the kinase and phosphatase reactions are irreversible, the rate constant for the exponential increase in 33P in the light chain is equal to the rate constant for the phosphatase reaction. Under activated conditions (pCa 4.5) at 20 degrees C, the incorporation of 33P into approximately 80% of the phosphorylated light chain is fit by a single exponential with a rate constant of 0.37 s-1. ATP usage due to phosphorylation and dephosphorylation of the light chain is about one-third of the suprabasal energy requirement. The high phosphatase rate constant suggests that dephosphorylation of the light chain is rapid enough to interact with and potentially modify the completion of the cross-bridge cycle.

Cross-bridge cycling at rest and during activation. Turnover of myosin-bound ADP in permeabilized smooth muscle.

Single turnover experiments were performed on myosin-bound ADP by measuring the time course of incorporation of [3H]ADP following rapid formation of [3H]ATP by photolysis of caged [3H]ATP. Permeabilized rabbit portal veins were incubated in a solution at 20 degrees C with 1 mM MgATP, 20 mM phosphocreatine, 1 mg/ml creatine phosphokinase, and containing [14C]ATP and high specific activity caged [3H]ATP. At variable times following a UV flash, the muscle was frozen, nucleotides were extracted, and the ratio 3H:14C in ADP was compared to that in ATP. At rest, the exchange of bound ADP occurred with a rate constant of 0.004 s-1. When the myosin light chain was about 80% thiophosphorylated, and the muscle was generating maximum isometric force, there appeared a fast phase of ADP exchange (44% of the total) which had a rate constant of 0.2 s-1. The change in rate of ADP exchange on myosin is sufficient to explain the measured increase in ATPase activity upon thiophosphorylation of the myosin light chain. A simple analysis of the data suggests that there is a 50-fold increase in the cycling rate of cross-bridges in the muscle upon phosphorylation under isometric conditions. The fraction of ADP exchanged at 10 s following photolytic release of [3H]ATP was found to be approximately linearly related to the degree of thiophosphorylation of the myosin light chain. This supports the idea that phosphorylation of the light chain causes the transition of myosin from the resting (slow ATPase) cycle into the activated (fast ATPase) cycle, and that the fraction of myosin in the fast cycle is directly determined by the degree of light chain phosphorylation. The data are also consistent with the cooperativity model described previously by Vyas et al.