Direct Effects of Ionizing Radiation on Macromolecules.

In the dry or frozen states, macromolecules are damaged directly by interactions with ionizing radiation. Since γ-rays and high-energy electrons randomly ionize orbital electrons in their path, larger molecules are more likely to suffer an interaction with these radiations. In each interaction, energy is transferred to the struck molecule, resulting in irreversibly broken covalent bonds. There is an extensive literature describing these radiation modifications in both synthetic and biopolymers. Although many different properties are measured, there emerges a similar picture of the nature of radiation damage that is common to all macromolecules. The techniques used in study of one species may be used to resolve questions raised in the other class of macromolecules.

A radiation target method for size determination of supercoiled plasmid DNA.

Supercoiled DNA plasmids were exposed in the frozen state to high-energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.

The molecular biology of Euglena gracilis. XV. Recovery from centrifugation-induced stratification.

The contents of Euglena gracilis cells can be separated in vivo by ultracentrifugation. Within the unbroken cell, each set of components forms a distinct layer according to their respective densities. The degree of segregation increases with both the g-force and the time of centrifugation, up to a maximum at 100,000 x g for 1 h, when six distinct strata can be observed. When returned to normal growth conditions, essentially all the cells return to the normal state and growth pattern. Greater g-forces or longer exposures do not alter the observable strata, but the ability of the cells to recover is diminished. Smaller g-forces result in less separation of cellular contents and all cells recover, even after 18 h of exposure. Euglena cells stratified at 100,000 x g for 1 h were returned to normal growth conditions; recovery was followed microscopically and by the rate of utilization of oxygen as well as that of the single carbon source. The cells recovered their normal state within 1 to 2 h, which is only a tenth of the normal doubling time. The mechanism for this recovery involves a natural process of change in cell shape caused by contraction and relaxation of the pellicle, a cell surface structure.

Direct radiation damage is confined to a single polypeptide in rabbit immunoglobulin G.

Frozen rabbit immunoglobulin G was exposed to high-energy electrons. The surviving polypeptide subunits were determined and analyzed by radiation target analysis. Each subunit was independently damaged by radiation whether or not they were bound by disulfide bridges to other subunits, demonstrating that in IgG radiation-deposited energy did not travel across disulfide bonds.

Effects of high-energy electrons and gamma rays directly on protein molecules.

High-energy electrons and gamma rays ionize molecules at random along their trajectories. In each event, chemical bonds are ruptured, releasing radiolytic products that diffuse away. A solution of macromolecules is mostly water whose principal radiation products are H(+) and OH(-). These can diffuse to and react with macromolecules; this indirect action of radiation is responsible for 99.9% of the damage to proteins. In frozen samples, the ionizations still occur randomly and water is still the principle molecular target, but diffusion of radiation products is limited to only a very small distance. At very low temperatures, essentially all the radiation damage to macromolecules is due to primary ionizations occurring directly in those molecules. Therefore, proteins in frozen solutions are only 10(-3) to 10(-4) as sensitive to radiation as in the liquid state. Every molecule that suffered a direct ionization is destroyed; the only surviving molecules are those that escaped ionization. The survival of frozen proteins after irradiation is a direct measure of the mass of the active structures and independent of the presence of other proteins.

Functional molecular mass of a vertebrate hyaluronan synthase as determined by radiation inactivation analysis.

Hyaluronan (HA), a linear polysaccharide composed of N-acetylglucosamine-glucuronic acid repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. The HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The prototypical vertebrate hyaluronan synthase, xlHAS1 (or DG42) from Xenopus laevis, is a 588-residue membrane protein. Recently, the streptococcal enzyme was found to function as a monomer of protein with approximately 16 lipid molecules. The vertebrate enzymes are larger than the streptococcal enzymes; based on the vertebrate HAS deduced amino acid sequence, two additional membrane-associated regions at the carboxyl terminus are predicted. We have utilized radiation inactivation to measure the target size of yeast-derived recombinant xlHAS1. The target size of HAS activity was confirmed using two internal standards. First, samples were spiked with glucose-6-phosphate dehydrogenase, an enzyme of known molecular weight. Second, parallel samples of native xlHAS1 and a xlHAS1-green fluorescent protein fusion (833 residues) were compared; substantial confidence was gained by using this novel internal standard. Our test also corroborated the basic tenets of radiation inactivation theory. We found that the vertebrate HAS protein functions catalytically as a monomer.

Radiation inactivation studies of hepatic sinusoidal reduced glutathione transport system.

Sinusoidal transport of reduced glutathione (GSH) is a carrier-mediated process. Perfused liver and isolated hepatocyte models revealed a low-affinity transporter with sigmoidal kinetics (K(m) approximately 3.2-12 mM), while studies with sinusoidal membrane vesicles (SMV) revealed a high-affinity unit (K(m) approximately 0.34 mM) besides a low-affinity one (K(m) approximately 3.5-7 mM). However, in SMV, both the high- and low-affinity units manifested Michaelis-Menten kinetics of GSH transport. We have now established the sigmoidicity of the low-affinity unit (K(m) approximately 9) in SMV, consistent with other models, while the high-affinity unit has been retained intact with Michaelis-Menten kinetics (K(m) approximately 0.13 mM). We capitalized on the negligible cross-contributions of the two units to total transport at the low and high ends of GSH concentrations and investigated their characteristics separately, using radiation inactivation, as we did in canalicular GSH transport (Am. J. Physiol. 274 (1998) G923-G930). We studied the functional sizes of the proteins that mediate high- and low-affinity GSH transport in SMV by inactivation of transport at low (trace and 0.02 mM) and high (25 and 50 mM) concentrations of GSH. The low-affinity unit in SMV was much less affected by radiation than in canalicular membrane vesicles (CMV). The target size of the low-affinity sinusoidal GSH transporter appeared to be considerably smaller than both the canalicular low- and high-affinity transporters. The high-affinity unit in SMV was markedly inactivated upon irradiation, revealing a single protein structure with a functional size of approximately 70 kDa. This size is indistinguishable from that of the high-affinity GSH transporter in CMV reported earlier.

Molecular mass and volume in radiation target theory.

Radiation target analysis is based on the action of ionizing radiation directly on macromolecules. Interactions of this radiation with the molecules leads to considerable structural damage and consequent loss of biological activity. The radiation sensitivity is dependent on the size of the macromolecules. There has been confusion and discrepancy as to whether the molecular mass or the molecular volume was the determinant factor in the sensitivity. Some proteins are known to change their hydrodynamic volume at low pH, and this characteristic can be utilized to compare the radiation sensitivities of these proteins in the two states. The results show that the radiation sensitivity of proteins depends on the mass of the molecule and is independent of the molecular volume/shape.

Radiation target analysis indicates that phenylalanine hydroxylase in rat liver extracts is a functional monomer.

The minimal enzymatically functional form of purified rat hepatic phenylalanine hydroxylase (PAH) is a dimer of identical subunits. Radiation target analysis of PAH revealed that the minimal enzymatically active form in crude extracts corresponds to the monomer. The 'negative regulation' properties of the tetrahydrobiopterin cofactor in both crude and pure samples implicates a large multimeric structure, minimally a tetramer of PAH subunits. Preincubation of the samples with phenylalanine prior to irradiation abolished this inhibition component without affecting the minimal functional unit target sizes of the enzyme in both preparations. The characteristics of rat hepatic PAH determined by studies of the purified enzyme in vitro may not completely represent the properties of PAH in vivo.

The active streptococcal hyaluronan synthases (HASs) contain a single HAS monomer and multiple cardiolipin molecules.

The functional sizes of the two streptococcal hyaluronan synthases (HASs) were determined by radiation inactivation analysis of isolated membranes. The native enzymes in membranes from Group A Streptococcus pyogenes HAS and Group C Streptococcus equisimilis HAS were compared with the recombinant proteins expressed in Escherichia coli membranes. Based on their amino acid sequences, the masses of these four proteins as monomers are approximately 48 kDa. In all cases, loss of enzyme activity was a simple single exponential function with increasing radiation dose. The functional sizes calculated from these data were identical for the four HASs at approximately 64 kDa. In contrast, the sizes of the proteins estimated by the loss of antibody reactivity on Western blots were essentially identical at 41 kDa for the four HAS species, consistently lower than the functional size by approximately 23 kDa. Matrix-assisted laser desorption time of flight mass spectrometry analysis of purified S. pyogenes HAS-H6 and S. equisimilis HAS-H6 gave masses that differed by <0.07% from the predicted monomer sizes, which confirms that neither protein is posttranslationally modified or covalently attached to another protein. Ongoing studies indicate that the purified HAS enzymes require cardiolipin (CL) for maximal activity and stability. When irradiated membranes were detergent solubilized and the extracts were incubated with exogenous CL, the residual level of HAS activity increased. Consequently, the calculated functional size decreased by approximately 23 kDa to the expected size of the HAS monomer. The approximately 23-kDa larger size of the functional HAS enzyme, compared with the HAS monomer, is due, therefore, to CL molecules. We propose that the active streptococcal HA synthases are monomers in complex with approximately 16 CL molecules.

Novel properties of hepatic canalicular reduced glutathione transport revealed by radiation inactivation.

Transport of GSH at the canalicular pole of hepatocytes occurs by a facilitative carrier and can account for approximately 50% of total hepatocyte GSH efflux. A low-affinity unit with sigmoidal kinetics accounts for 90% of canalicular transport at physiological GSH concentrations. A low-capacity transporter with high affinity for GSH has also been reported. It is not known whether the same or different proteins mediate low- and high-affinity GSH transport, although they do differ in inhibitor specificity. The bile of rats with a mutation in the canalicular multispecific organic anion transporter (cMOAT or MRP-2, a 170-kDa protein) is deficient in GSH, implying that cMOAT may transport GSH. However, transport of GSH in canalicular membrane vesicles (CMV) from these mutant rats remains intact. We examined the functional size of the two kinetic components of GSH transport by radiation inactivation of GSH uptake in rat hepatic CMV. High-affinity transport of GSH was inactivated as a single exponential function of radiation dose, yielding a functional size of approximately 70 kDa. In contrast, low-affinity canalicular GSH transport exhibited a complex biexponential response to irradiation, characterized by an initial increase followed by a decrease in GSH transport. Inactivation analysis yielded a approximately 76-kDa size for the low-affinity transporter. The complex inactivation indicated that the low-affinity transporter is associated with a larger protein of approximately 141 kDa, which masked approximately 80% of the potential transport activity in CMV. Additional studies, using inactivation of leukotriene C4 transport, yielded a functional size of approximately 302 kDa for cMOAT, indicating that it functions as a dimer.

Radiation effects on the native structure of proteins: fragmentation without dissociation.

Several proteins (avidin, carboxypeptidase B, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, maltase, and peroxidase) composed of one to six subunits were irradiated in the frozen state. Each irradiated protein was examined by size-exclusion chromatography (SEC) and by denaturing gel electrophoresis (SDS-PAGE). All these proteins eluted from SEC as a single peak even though SDS-PAGE showed cleavage of the polypeptide backbone of the monomers. Thus, fragmentation of the subunits did not result in dissociation of the oligomeric structure.

Size of the catalytically active unit of rat hepatic carboxylester lipase in the presence and absence of bile salt.

Carboxylester lipase (CEL) catalyzes the hydrolysis of cholesteryl esters, retinyl esters, and triacylglycerols. CEL monomer has a MW of approximately 70000. Hydrolysis of these esters is stimulated by millimolar trihydroxy bile salts such as cholate, that also induce aggregation. Liver cytosols from 12 rats were frozen and irradiated at -135 degrees C with high energy electrons. In several experiments, paired samples of cytosol were adjusted to 20 mM cholate before irradiation. All samples were assayed for CEL using cholesteryl oleate as substrate. In untreated cytosols, CEL activity surviving radiation exposure could be fit to a single exponential function, the slope of which yielded a target size of 91 +/- 18 kDa. In a subset of these cytosols irradiated in the presence of cholate the calculated target size was 100 +/- 19 kDa, a value indistinguishable from that obtained for untreated cytosols. Some samples were also assayed using retinyl palmitate and triolein as substrates. With retinyl palmitate the mean target sizes were 96 and 108 kDa in the absence and presence of cholate, respectively, approximately the same as those observed when using cholesteryl oleate. When triolein was used as substrate the target sizes in the absence of cholate were smaller than for the other two esters (67 +/- 18 kDa) and closer to the known monomer molecular weight, but again cholate had no significant effect on this size. The structure responsible for CEL activity contains no more than one 70000 MW monomer and the results show that cholate-induced oligomerization is not required for catalytic activity.

Structure-function relationships in Escherichia coli transcription termination protein Rho revealed by radiation target analysis.

High-energy electrons were used to measure the target sizes for inactivation of the RNA-dependent ATPase activity of Escherichia coli transcription termination factor Rho, for its ATP binding ability, and for its physical destruction. SDS-PAGE analysis of irradiated samples indicated that the target size for polypeptide destruction in the homohexameric enzyme is the dimer, indicating that energy transfer must occur from a hit subunit to one other subunit, although the subunits are not known to be linked by any covalent bonds. The ATP binding ability of Rho also inactivates as a dimer, a result that is consistent with the physical destruction target size. However, a single subunit as the ATP binding entity is not excluded. The RNA-dependent ATPase activity of Rho inactivates with the apparent target size of trimer to tetramer, indicating that interactions among the subunits of Rho are required for ATP hydrolysis. Rho hexamers are known to exchange subunits, although the identity of the exchanging unit is not known. Models in which this property of Rho is taken into account indicate that the closest fit to the experimental data is for an ATPase target size of a hexamer with dimers as the exchanging units, consistent with earlier chemical inactivation studies.

Human hepatic lipase subunit structure determination.

Chinese hamster ovary cells were stably transfected with a human hepatic lipase (HL) cDNA. The recombinant enzyme was purified from culture medium in milligram quantities and shown to have a molecular weight, specific activity, and heparin affinity equivalent to HL present in human post-heparin plasma. The techniques of intensity light scattering, sedimentation equilibrium, and radiation inactivation were employed to assess the subunit structure of HL. For intensity light scattering, purified enzyme was subjected to size exclusion chromatography coupled to three detectors in series: an ultraviolet absorbance monitor, a differential refractometer, and a light scattering photometer. The polypeptide molecular weight (without carbohydrate contributions) was calculated using the measurements from the three detectors combined with the extinction coefficient of human HL. A single protein peak containing HL activity was identified and calculated to have a molecular mass of 107,000 in excellent agreement with the expected value for a dimer of HL (106.8 kDa). In addition, sedimentation equilibrium studies revealed that HL had a molecular mass (with carbohydrate contributions) of 121 kDa. Finally, to determine the smallest structural unit required for lipolytic activity, HL was subjected to radiation inactivation. Purified HL was exposed to various doses of high energy electrons at -135 degrees C; lipase activity decreased as a single exponential function of the radiation dose to less than 0.01% remaining activity. The target size of functional HL was calculated to be 109 kDa, whereas the size of the structural unit was determined to be 63 kDa. These data indicate that two HL monomer subunits are required for lipolytic activity, consistent with an HL homodimer. A model for active dimeric hepatic lipase is presented with implications for physiological function.

High-energy electron irradiation of proteins and nucleic acids: collisional stopping power and average energy loss.

Inactivation of proteins due to the direct action of ionizing radiation and the electron energy loss spectra of organic materials indicate that an average of 60-66 eV of energy is lost from high energy electrons in each inelastic collision with target molecules. The average energy loss per inelastic collision with high energy electrons in solid, carbon-based materials, proteins and nucleic acids is calculated from mass collisional stopping powers and empirical total inelastic cross-sections. Bragg's Additivity Law is used for the calculation of the mean excitation energy of molecules. For simple organic compounds, the calculated average energy loss is close to that obtained by direct observation of the energy loss suffered by electrons as they pass through thin films of organic material. The density effect correction for the rate of energy loss, important in the more complex case of proteins irradiated with 10 MeV electrons, is determined using the comparable mass collisional stopping power of water and proteins. In this manner, a value is obtained for the average energy per inelastic collision of high energy electrons with proteins, which is similar to the average energy per inactivating event of proteins. Analogous calculations for nucleic acids are also presented.