Target Size Analysis Research Articles

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.

Target Size Analysis by Radiation Inactivation: The Use of Free Radical Scavengers

Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.

Structural studies of the H+/oligopeptide transport system from rabbit small intestine

A 127-kDa protein was identified as a component of the H+/oligopeptide transport system in brush-border membrane vesicles from rabbit small intestine by photoaffinity labeling with [3H]cephalexin and further photoreactive β-lactam antibiotics and dipeptides. Reconstitution of stereospecific transport activity revealed the involvement of the 127-kDa protein in H+-dependent transport of oligopeptides and orally active α-amino-β-lactam antibiotics (Kramer et al., Eur. J. Biochem. 204 (1992) 923–930). H+-Dependent transport activity was found in all segments of the small intestine concomitantly with the specific labeling of the 127-kDa protein. By enzymatic deglycosylation, fragments of Mr 116 and 95 kDa were obtained from the 127-kDa protein with endoglucosidase F and N-glycanase, whereas with endoglucosidase H, a fragment of Mr 116 kDa was formed. These findings indicate that the photolabeled 127-kDa protein is a microheterogenous glycoprotein. Surprisingly, it was found that the solubilized and purified 127-kDa protein showed enzymatic sucrase and isomaltase activity. Inhibition of the glucosidase activities with the glucosidase inhibitor HOE 120 influenced neither H+/oligopeptide transport nor photoaffinity labeling of the 127-kDa protein. With polyclonal antibodies raised against the purified 127-kDa protein, a coprecipitation of sucrase activity and the photolabeled 127-kDa β-lactam antibiotic binding protein occurred. Target size analysis revealed a functional molecular mass of 165±17 kDa for photoaffinity labeling of the 127-kDa protein, suggesting a homo- or heterodimeric functional structure of the 127-kDa protein in the brush-border membrane. These findings indicate that the H+/oligopeptide binding protein of Mr 127 000 is closely associated with the sucrase/isomaltase complex in the enterocyte brush-border membrane.

Target size analysis of an avermectin binding site from Drosophila melanogaster

A high-affinity avermectin binding site from Drosophila melanogaster head membranes was subjected to target size analysis by radiation inactivation in order to determine the functional unit size. Using the [ 3H]ivermectin binding assay to assess ligand binding activity, the target size was determined to be 44.3±3.9 kDa. This result suggests that the size of the functional unit required for high-affinity ligand binding is a monomer. The membrane-associated acetylcholinesterase present in the Drosophila head membranes was also analyzed by radiation inactivation and served as a control. By monitoring enzymatic activity, the functional unit size of the Drosophila acetylcholinesterase was determined to be 70±9.7 kDa. This corresponds to the molecular weight of a dimer composed of a 55 kDa subunit and a 16–18 kDa subunit.

Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation.

Vacuolar proton-pyrophosphatase (H(+)-PPase) of mung bean seedlings contains a single kind of polypeptide with a molecular mass of approx. 73 kDa. However, in this study, a molecular mass of approx. 140 kDa was obtained for the purified vacuolar H(+)-PPase by size-exclusion gel-filtration chromatography, suggesting that the solubilized form of this enzyme is a dimer. Radiation inactivation analysis of tonoplast vesicles yielded functional masses of 141.5 +/- 10.8 and 158.4 +/- 19.5 kDa for PP1 hydrolysis activity and its supported proton translocation respectively. These results confirmed the in situ dimeric structure of the membrane-bound H(+)-PPase of plant vacuoles. Further target-size analysis showed that the functional unit of purified vacuolar H(+)-PPase was 71.1 +/- 6.7 kDa, indicating that only one subunit of the purified dimeric complex would sufficiently display its enzymic reaction. Moreover, in the presence of valinomycin and KCl, the functional size of membrane-bound H(+)-PPase was decreased to approx. 63.4 +/- 6.3 kDa. A working model was proposed to elucidate the structure of native H(+)-PPase on vacuolar membrane as a functional dimer. Factors that would disturb the membrane, e.g. membrane solubilization and the addition of valinomycin and KCl, may induce an alteration in its enzyme structure, subsequently resulting in a different functional size.

Estimation by radiation inactivation of the minimum functional size of acrosome-reaction-including substance (ARIS) in the starfish,Asterias amurensis

In the starfish Asterias amurensis, the jelly coat of the eggs contains a glycoprotein essential for the induction of the acrosome reaction in homologous spermatozoa that is termed the acrosome-reaction-inducing substance (ARIS). ARIS is a highly sulphated and fucose-rich glycoprotein of extremely high molecular mass (> 10(4) kDa). ARIS was irradiated with high-energy electrons in order to estimate the minimum size required for its biological activity. The minimum functional unit or target size of ARIS was estimated to be c. 14 kDa by target size analysis. ARIS was significantly disintegrated by the irradiation, yet the total sugar content was not apparently reduced. The binding of 125I-labelled ARIS to spermatozoa competed with that of irradiated ARIS, although the affinity of ARIS was much reduced after irradiation.

Inactivation of secretory phospholipase A2 by ionizing radiation

The extracellular phospholipase A2s (PLA2) from cobra venom, rattlesnake venom, and porcine pancreas were analyzed by radiation inactivation to determine their functional aggregation states. The analysis was performed in the presence of the protein transferrin at two different concentrations of PLA2: 5 micrograms/ml. The small size of these proteins necessitated the use of high radiation dosages. The catalytic activity of all samples decreased as a single exponential as a function of radiation dosage, to > 97% inactivation. Target size analysis of these curves yielded sizes corresponding to dimers for all three PLA2s, indicating that all three enzymes exist as dimers or larger aggregates under the conditions studied. An analysis of the amount of intact protein remaining by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the loss of protein also followed a dimeric size for all three PLA2s. The loss of protein as a dimer indicates that transfer of radiation energy is occurring between polypeptides.

Target size analysis of the peptide/H +-symporter in kidney brush-border membranes

The apparent functional molecular mass of the kidney peptide/H +-symporter was determined by radiation inactivation in brush-border membrane vesicles (BBMV) of rat kidney cortex. Purified BBMV were irradiated at low temperatures with high energy electrons generated by a 10-MeV linear accelerator at doses from 0 to 30 megarads. Uptake studies were performed with [ 3H]cefadroxil, a β-lactam antibiotic which serves as a substrate for the kidney peptide/H +-symporter. Inhibition of influx of [ 3H]cefadroxil into BBMV was used to determine the functional molecular mass of the transporter. Additionally, direct photoaffinity labeling of the transport-and/or binding proteins for [ 3H]cefadroxil in control and irradiated BBMV was performed to determine the molecular mass of the putative transporter by SDS-polyacrylamide gel electrophoresis. Initial rates of pH-gradient dependent uptake of [ 3H]cefadroxil decreased progressively as a function of radiation dose. The apparent radiation inactivation size (RIS) of the transport function was found to be 414 ± 16 kDa. Direct photoaffinity labeling yielded labeled membrane proteins with apparent molecular masses of 130 kDa and 105 kDa, respectively. The proteins displayed different labeling characteristics with respect to incubation time, specificity and the response to irradiation. It appears that only a 105 kDa protein is directly involved in transport function since (a) only it showed a specific pH gradient dependent labeling pattern and (b) the covalent incorporation of [ 3H]cefadroxil into this protein decreased parallel to the loss of transport function in irradiated BBMV. The peptide/H +-symporter in kidney brush-border membranes therefore appears to have a monomer mass of 105 kDa and may function in an oligomeric arrangement.

Detection of oligomeric and monomeric forms of P-glycoprotein in multidrug resistant cells.

P-glycoprotein (P-gp) is thought to function as a drug efflux pump in multidrug resistant (MDR) cells. The functional form of P-gp in its native state is not known. Previous results from radiation target size analysis have suggested that P-gp occurs as dimers in MDR cell plasma membranes [Boscoboinik et al. (1990) Biochim. Biophys. Acta 1027, 225-228]. In this study, we used sucrose gradient velocity sedimentation to determine if P-gp oligomers could be retrieved from detergent extracts of hamster and human MDR cell lines. The proportion of P-gp recovered as higher order oligomers was dependent on the detergents used for solubilization of the cells. When a detergent such as CHAPS was used, 50% or more of the P-gp sedimented as higher order oligomers. In contrast, in the presence of SDS, only monomers were retrieved, but naturally occurring oligomers could be preserved if the cells were treated with a cross-linker prior to detergent solubilization. The oligomers and monomers were both able to bind the photoactive analog of ATP (8-azido[alpha-32P]ATP) or the drug [3H]azidopine in membrane preparations. P-gp is a phosphoprotein, and its phosphorylated state is thought to be important for function. When MDR cells were labeled with [32P]orthophosphate in vivo, we observed that the monomer and dimer were more highly phosphorylated than the larger oligomers, suggesting that these different forms of P-gp may be functionally distinct. The assembly of oligomers appears to occur in an early bisynthetic compartment, and asparagine-linked glycosylation is not required for their formation. Our findings indicate that oligomers of P-gp exist in MDR cells and raise the possibility that the dynamics of oligomer formation and dissociation may be important in the mechanism of action of P-gp.

Function and presumed molecular structure of Na+-D-glucose cotransport systems

Functional characterization of Na(+)-D-glucose cotransport in intestine and kidney indicates the existence of heterogeneous Na(+)-D-glucose cotransport systems. Target size analysis of the transporting unit and model analysis of substrate binding have been performed and proteins have been cloned which mediate (SGLT1) and modulate (RS1) the expression of Na(+)-D-glucose cotransport. The experiments support the hypothesis that functional Na(+)-D-glucose cotransport systems in mammals are composed of two SGLT1-type subunits and may contain one or two RS1-type proteins. SGLT1 contains up to twelve membrane-spanning alpha-helices, whereas RS1 is a hydrophilic extracellular protein which is anchored in the brush-border membrane by a hydrophobic alpha-helix at the C-terminus. SGLT1 alone is able to translocate glucose together with sodium; however, RS1 increases the Vmax of transport expressed by SGLT1. In addition, the biphasic glucose dependence of transport, which is typical for kidney and has been often observed in intestine, was only obtained after coexpression of SGLT1 and RS1.

Effect of Environmental Conditions on Radiation Target Size Analyses

Target size determinations from radiation inactivation of proteins is dependent on the physical and chemical environment of the sample during radiation exposure. Effects of temperature and physical state have already been described. Buffers, the effects of protein concentration, and the addition of small molecules are examined for several enzymes. Phosphate buffer is found to have major effects on the rate of inactivation of certain, but not all, proteins. The amount of protein in irradiated samples is significant for all enzymes studied; the nature of the specific protein used is unimportant. Neither sucrose nor other glycitols could substitute for protein in target size determinations. Certain small molecules, especially cysteamine, were effective in sparing the need for high protein levels in radiation inactivation studies of four enzyme systems.

Structure of Na-<i>D</i>-Glucose Cotransport Systems

Functional characterization of Na⁺-<i>D</i>-glucose cotransport and the analysis of genetic diseases indicate heterogeneous Na⁺-<i>D</i>-glucose cotransport systems in the intestine and in various regions of the kidney. Target size analysis of the transporting unit and model analysis of phlorizin binding to the transporter suggest that the transport systems have a molecular mass around 300,000 daltons and contain two coexisting transport sites. Two types of proteins are thought to be components of Na⁺-<i>D</i>-glucose cotransport systems: A membrane protein with up to 12 membrane-spanning α-helices (SGLT1) which has been cloned from intestine and is also found in kidney, and a membrane-associated extracellular protein (RS1) which has been cloned from kidney and is also present in intestine. RS1 is anchored in the brush-border membrane by a hydrophobic α-helix at the C-terminus. SGLT1 alone is able to translocate glucose together with sodium; however, RS1 increases the V_max of the uptake expressed by SGLT1. In addition, the biphasic glucose dependence of transport which is typical for kidney and has also been observed in intestine, was only obtained after the coexpression of both SGLT1 and RS1. The data support the hypothesis that Na⁺-<i>D</i>-glucose cotransport systems are composed of two SGLT1-type and one or two RS1-type proteins.

Pseudomonas aeruginosa cytotoxin‐binding protein in rabbit erythrocyte membranes

Rabbit erythrocyte membrane glycosylated 28-kDa protein was investigated in the membrane-bound as well as in the soluble state on an example of a Pseudomonas aeruginosa cytotoxin-binding component. When membranes were treated with trypsin/N-glycosidase F, a 13.5-kDa-binding active peptide residue is obtained as revealed by a ligand-blot technique after separation by SDS/PAGE under reducing conditions and electrophoretic transfer to nitrocellulose. Target-size analysis of intact membranes by radiation inactivation using 2-450 kGy gave a value of 29, 40 and 60 kDa for the binding-protein structure. This suggests that the native form of the binding peptide is associated as an oligomer. As seen in ligand-blot technique, 125I-cytotoxin binds with high affinity to water-channel integral protein CHIP28 from human erythrocyte membranes. The 20 N-terminal amino acids of the deglycosylated rabbit cytotoxin-binding protein show high similarity to transmembrane channel-like proteins.

Chronic opioid treatment may uncouple opioid receptors and G-proteins: evidence from radiation inactivation analysis

Radiation inactivation (target size analysis) was used in this study to determine whether uncoupling of opioid receptor and G-protein is a contributing mechanism to opioid tolerance. Male Sprague-Dawley rats (160–260 g) were rendered tolerant to morphine or [D-Ala 2, D-Leu 5]enkephalin (DADLE) by multiple i.p. or i.c.v. injections twice a day for 6 or 5 days. Control rats were injected with saline instead of opioids. The animals were killed, the midbrains excised and pooled together for each group. The washed P 2 membranes were suspended in buffer and irradiated with 1–10 Mrad doses of 60Co irradiation, following which μ- or δ-opioid receptor binding activity of each sample was assayed. The molecular weight of the receptor was calculated from a standard irradiation curve constructed using several enzyme markers of known molecular weight. We found that the functional molecular size of μ-opioid receptor significantly decreased from 349 kDa to 228 kDa after 6 days of chronic morphine treatment, while, the molecular size of δ-opioid receptor decreased from 303 kDa to 223 kDa after 5 days of chronic DADLE treatment. These results are consistent with the uncoupling of opioid receptor from G-protein during chronic opioid treatment.

Identification and target-size analysis of the leukotriene D 4 receptor in the human THP-1 cell line

The human acute monocytic leukemia cell line THP-1 has been identified, by radioligand binding, as expressing the leukotriene D 4 receptor at a high level (4000 binding sites per cell), without the need for further cell differentiation. [ 3H]Leukotriene D 4-specific binding to THP-1 cell membranes was of high affinity ( K D = 0.47 nM) and saturable, enhanced by divalent cations but inhibited by both monovalent cations and non-hydrolyzable GTP analogs. The cysteinyl leukotrienes competed for [ 3H]leukotriene D 4-specific binding with the following rank order of potency: leukotriene D 4 ⪢ leukotriene E 4 〉 leukotriene C 4 In addition, leukotriene D 4-receptor antagonists from two structural classes, the quinolines MK-571 and L-697,008, and the indole ICI 204,219, displayed nanomolar potency in [ 3H]leukotriene D 4 competition assays. These data show that [ 3H]leukotriene D 4-specific binding to THP-1 cell membranes fulfils the criteria for binding to a leukotriene D 4 receptor regulated through interaction with a G protein. Several novel features of the THP-1 leukotriene D 4 receptor were investigated. Culture of THP-1 cells in the presence of tunicamycin, an inhibitor of N-glycosylation, resulted in a 6-fold decrease in the number of detectable [ 3H]leukotriene D 4-specific binding sites. Target-size analysis by radiation inactivation estimated a molecular mass of 65 kDa for the [ 3H]leukotriene D 4 specific binding site(s) present in THP-1 cell membranes. Together, these results suggest that the human THP-1 cell leukotriene D 4 receptor is a glycosylated protein with a molecular mass of approx. 65 kDa within the membrane environment.

Photoaffinity labelling and radiation inactivation of the leukotriene B 4 receptor in human myeloid cells

The leukotriene (LT) B 4 receptor has been characterized in the human monocyte leukaemia THP-1 cell line. Scatchard analysis of [ 3H]LTB 4 specific binding to THP-1 cell membranes revealed a single population of high affinity (K D = 56 pM) and saturable (2000 receptors / cell) binding sites. [ 3H]LTB 4 specific binding was enhanced by divalent cations, but inhibited by both monovalent cations and a non-hydrolysable GTP analogue. Treatment with GTP analogue resulted in a concentration-dependent reduction in the number of high affinity binding sites, accompanied by the appearance of an equal number of binding sites of lower affinity (K D = 1250 pM). In contrast, Scatchard analysis with human polymorphonuclear leukocyte (PMN) membranes consistently revealed two populations of LTB 4 receptors (K D = 48 pM and 270 pM). Treatment with GTP analogue, however, converted all these detectable binding sites to the lower affinity state. These data suggest that the LTB 4 receptor in both THP-1 cell and PMN membranes exists in interconverting affinity states modulated by G-protein coupling. The similarity between the LTB 4 receptors present in these two cell types was also substantiated by target-size analysis by radiation inactivation, which estimated a comparable molecular mass of 56.5 kDa and 52.8 kDa for the THP-1 cell and PMN LTB 4 receptors, respectively. Finally, the presence of a single LTB 4 receptor in PMN was demonstrated by direct photolabelling. Irradiation of frozen [ 3H]LTB 4 equilibrium binding assay incubations resulted in complete photolysis of [ 3H]LTB 4. Subsequent resolution of the tritiated PMN proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed one major radioactive peak migrating with an apparent molecular weight of 61,000. This peak was identified as the LTB 4 receptor since radiolabelling could be completely inhibited by the presence of excess unlabelled LTB 4 or the LTB 4-receptor antagonist, L-662,328. Photolabelling was also partially inhibited by pretreatment with GTP analogue, consistent with G-protein uncoupling reagents reducing receptor affinity without complete inhibition. In summary, the LTB 4 receptor identified in human myeloid cells is a G-protein coupled receptor with interconvertible high and low affinity states, having a molecular mass of 53–61 kDa.

A 30 kDa functional size for the erythrocyte water channel determined in situ by radiation inactivation

The functional unit size of the water channel in rabbit erythrocytes was assessed using target size analysis following radiation inactivation. Using Radiochromic nylon dosimetry, accurate values of accumulated dose yielded an absolute target analysis, leading to direct determination of molecular size. The erythrocyte water channel functional size was shown to be 30 kDa, and is identical to the size found in rat renal proximal tubule brush border membranes (1), suggesting close homology of these two water channels. The result suggests that the 28 kDa channel-like intrinsic protein (CHIP28) recently isolated from human erythrocytes and proximal tubule (2), which is believed to form water channels of oligomeric construction may have a functional water channel activity in monomeric form.

Target size analysis of prostaglandin endoperoxide synthase. Radiation inactivation of both cyclooxygenase and peroxidase correlated with the monomer of 72 kDa.

To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono-exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.

Radiation target sizes of the Na,K-ATPase and p-aminohippurate transport system in the basolateral membrane of renal proximal tubule

Basolateral membrane vesicles made from rabbit kidney proximal tubules were frozen and irradiated with a high energy electron beam and the effects of irradiation on Na,K-ATPase activity, p-aminohippurate (PAH) transport, the membrane diffusion barrier and vesicle volume were measured. The vesicle volume and diffusion barrier were not significantly changed by radiation exposure. Na,K-ATPase activity was inactivated as a simple exponential function of radiation dose. Target size analysis of the data yielded a molecular size of 267 ± 17 kDa, consistent with its existence as a (αβ) 2 dimer. The carrier-mediated PAH uptake by basolateral membrane vesicles was also inactivated as a function of radiation dose. A target molecular size of 74 ± 16 kDa was calculated for the PAH transport system. This study is the first measurement of the functional size of the organic acid transport system based directly on flux measurements.

Target size analysis of estrogen receptor in cultured intact cells: Change in receptor structure between subconfluency and superconfluency in culture

MCF-7 human breast cancer cells were submitted to the tritiated antiestrogen tamoxifen aziridine, frozen at — 170°C, stored and irradiated at —78°C in a calibrated Gammacell 60Co irradiator. A three-step protein extraction procedure provided protein samples for the determination of the target size (TS) of the covalently labelled estrogen receptor (ER). From the TS it is shown that ER bound to an antiestrogen was, in whole cells, part of a 265 kDa polypeptide structure if measured in MCF-7 cells at subconfluency, or of a 360 kDa species in superconfluent cells.