Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase.

Although normal intracellular levels of arginine are well above the K(m), and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox," has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.

Stimulation of receptor-mediated nitric oxide production by vanadate.

Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore- (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to approximately 125 kDa and 2 minor bands corresponding to approximately 105 and 75 kDa after treatment with vanadate/Bk. No tyrosine phosphorylation of eNOS after treatment with vanadate/Bk was observed. Geldanamycin, an inhibitor of heat shock protein 90, also inhibited the enhancement of NO production by vanadate/Bk or vanadate/A23187, and there was an increase in the amount of heat shock protein 90 that coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These results show that there is a clear link between tyrosine phosphorylation and stimulation of eNO production, which does not appear to involve direct modification of eNOS, changes in eNOS levels, or compartmentation, but rather appears to be due to changes in proteins associating with eNOS, thereby enhancing the state of activation of eNOS.

1H and 13C NMR studies of a truncated heme domain from Chlorella vulgaris nitrate reductase: signal assignment of the heme moiety.

A water soluble truncated heme domain (a tetramer of MW = 45 kDa) of the tetrameric nitrate reductase complex from the green alga Chlorella vulgaris has been overexpressed and purified. This truncated heme domain with four identical subunits has a high redox potential (midpoint potential E1/2 = +16 mV) as compared with other heme-containing flavoproteins. We have undertaken a determination of the detailed configuration of the heme moiety in order to understand the unique electrochemical property of the heme moiety of this enzyme. We report here the study of the heme prosthetic group of the truncated heme domain by the use of 2D 1H and 13C NMR techniques. A complete signal assignment of the heme has been achieved. Our observations suggest that the heme configuration is similar to that of the crystal structure of the membrane-bound bovine liver cytochrome b5.

Calmodulin promotes dimerization of the oxygenase domain of human endothelial nitric-oxide synthase.

The active form of endothelial nitric-oxide synthase (eNOS) is a homodimer. The activity of the enzyme is regulated in vivo by calcium signaling involving the binding of calmodulin (CAM), which triggers the activation of eNOS. We have examined the possible role of calcium-mediated CAM binding in promoting dimerization of eNOS through the oxygenase domain of the enzyme. A recombinant form of the oxygenase domain of human eNOS was expressed in a prokaryotic expression system. This recombinant domain contains the catalytic cytochrome P-450 site for arginine oxidation by molecular oxygen as well as the binding sites for tetrahydrobiopterin and Ca2+-CAM but lacks the reductase domain and associated FAD, FMN, and NADPH binding sites. Binding of Ca2+-CAM caused an association of monomeric eNOS oxygenase domain as determined by changes in fluorescence, both intrinsic and extrinsic, and by gel filtration, chemical cross-linking, and particle-sizing. Dimerization of the domain was not dependent on the presence of the substrate, arginine, or the cofactor, tetrahydrobiopterin. A truncated form of the eNOS oxygenase domain lacking the Ca2+-CAM binding region did not undergo self-association to form dimers. These results show that the eNOS reductase domain is not required for Ca2+-CAM-induced dimerization of eNOS and suggest that this dimerization may be a primary event in the activation of eNOS by Ca2+.

Cloning and characterization of the nitrate reductase-encoding gene from Chlorella vulgaris: structure and identification of transcription start points and initiator sequences.

The reduction of nitrate to nitrite catalyzed by nitrate reductase (NR) is considered to be the rate-limiting and regulated step of nitrate assimilation, a major metabolic pathway occurring in a wide range of organisms which in turn supply the nutritional nitrogen requirements for other forms of life. Chlorella vulgaris NR mRNA levels are very responsive to changes in nitrogen source. In the presence of ammonia as the sole nitrogen source, under repressed conditions, NR mRNA is undetectable. Under inducing conditions, the removal of ammonia and addition of nitrate, rapid NR mRNA synthesis occurs. We are studying the elements involved in regulating the expression of this important gene. Two overlapping genomic clones (NRS1 and NR5') were isolated from a cosmid library. The two clones were sequenced and their sequences were aligned with that of a full-length NR cDNA. The gene is approximately 8 kb long and consists of 19 exons and 18 introns. Unlike NR isolated from other species, the exons which code for the functional domains of C. vulgaris are separated by introns. Two transcription start points (tsp) were identified and each is surrounded by potential initiator sequences. No TATA, CAAT or GC-rich promoter elements were located. A time course of NR induction revealed that while transcription initiation from one tsp remains at a constant level from the point of induction through steady state, the level of initiation from another tsp is high upon induction, but decreases as steady state is attained.

Nitric oxide release from resting human platelets.

In this study, we have investigated the release of nitric oxide from resting human platelets. Nitric oxide was detected and quantitated by either measuring the conversion of oxy-hemoglobin to met-hemoglobin or generation of nitrite and nitrate by the cells. Nitric oxide was released from both intact resting platelets and platelets activated by collagen. Nitric oxide release was proportional to platelet concentration, and was equivalent to approximately 4.5 +/- 0.6 pmol (or 2.8 +/- 0.3 pmol in the presence of prostaglandin I2) and 11.2 +/- 1.3 pmol nitric oxide released per minute per 10(8) cells at 37 degrees C for resting platelets and platelets activated by collagen, respectively. The generation of nitric oxide by resting platelets was linear with respect to time over a two hour period, while the release of nitric oxide from platelets following activation was transient and was linear for only the first 10 min, after which it slowed to completion at approximately 30 min. The release of nitric oxide was it slowed to completion at approximately 30 min. The release of nitric oxide was stimulated by L-arginine, but was inhibited by L-nitro-arginine methyl ester (L-NAME). The inhibitory effect of L-NAME could be reversed by addition of L-arginine. The release of nitric oxide from platelets was also partially inhibited by prostaglandin I2, prostaglandin E1, aspirin and EDTA. The amount of nitric oxide released from resting platelets compared with that released from endothelial cells suggests that platelet-derived nitric oxide may play a significant role in the maintenance of vascular tone and blood flow.

Characterization of a maize root proteinase.

The major proteinase in maize (Zea mays) roots behaves as a serine endopeptidase. A possible physiological role of this enzyme could be in the turnover of nitrate reductase (NR) and, as such, it could be of great importance in regulating the assimilation of nitrate. The objective of this research was to elucidate the specificity and uniqueness of maize root proteinase. When bovine serum albumin and an NR purified from Chlorella vulgaris were used as substrates, the maize root proteinase exhibited a preference for cleavages such that the amino acid on the amino side of the scissile bond was alanine. This information was established by microsequence analysis of the N termini of proteolytic fragments, and carboxypeptidase Y analysis of the C termini of proteolytic fragments of substrates hydrolyzed by the proteinase. Cleavage occurred at the sequence Ala/Ala-Ala-Ala-Pro-Glu in Chlorella NR, and at the sequence Ala-Asp-Glu-Ser-His-Ala-Gln in bovine serum albumin. When bovine serum albumin was the substrate, the maize root proteinase yielded a peptide map that is unique relative to those created with the other serine endopeptidases elastase, trypsin, or chymotrypsin. Based on our data, the maize root proteinase appears to cleave peptide bonds at the carboxy side of alanine. Because of its specificity, it should have useful applications in protein chemistry.

Expression and characterization of the heme-binding domain of Chlorella nitrate reductase.

A recombinant protein corresponding to the putative heme-binding domain of assimilatory NADH:nitrate reductase from Chlorella vulgaris has been expressed and purified from transformed Escherichia coli BL21 cells. The recombinant protein, exhibited a subunit molecular mass of approximately 10 kDa with a N-terminal sequence beginning with the residues PAGA in agreement with that predicted by cDNA analysis. The UV-visible spectrum of the protein confirmed the incorporation of heme with maxima at 413 nm and 423, 528, and 557 nm for the oxidized and reduced forms, respectively. Circular dichroism spectra indicated the environment of the heme chromophore was very similar to that of the native enzyme. Potentiometric titrations of the recombinant heme domain yielded a midpoint potential of +16 mV (n = 1, pH 7), substantially higher than the values of -160 mV obtained for the native enzyme and -28 mV obtained for a previously expressed recombinant heme domain that contained part of the Mo-pterin domain. These results indicate that portions of the amino acid sequence that are involved in the formation of the Mo-pterin domain of Chlorella nitrate reductase influence the redox potential of the heme prosthetic group.

Electrochemical and kinetic analysis of electron-transfer reactions of Chlorella nitrate reductase.

Assimilatory nitrate reductase (NR) from Chlorella is homotetrameric, each subunit containing FAD, heme, and Mo-pterin in a 1:1:1 stoichiometry. Measurements of NR activity and steady-state reduction of the heme component under conditions of NADH limitation or competitive inhibition by nitrite suggested intramolecular electron transfer between heme and Mo-pterin was a rate-limiting step and provided evidence that heme is an obligate intermediate in the transfer of electrons between FAD and Mo-pterin. In addition to the physiological substrates NADH and nitrate, various redox mediators undergo reactions with one or more of the prosthetic groups. These reactions are coupled by NR to NADH oxidation or nitrate reduction. To test whether intramolecular redox reactions of NR were rate-determining, rate constants for redox reactions between NR and several chemically diverse mediators were measured by cyclic voltammetry in the presence of NADH or nitrate. Reduction of ferrocenecarboxylic acid, dichlorophenolindophenol, and cytochrome c by NADH-reduced NR was coupled to reoxidation at a glassy carbon electrode (ferrocene and dichlorophenolindophenol) or at a bis(4-pyridyl) disulfide modified gold electrode (cytochrome c), yielding rate constants of 10.5 x 10(6), 1.7 x 10(6), and 2.7 x 10(6) M-1 s-1, respectively, at pH 7. Kinetics were consistent with a second-order reaction, implying that intramolecular heme reduction by NADH and endogenous FAD was not limiting. In contrast, reduction of methyl viologen and diquat at a glassy carbon electrode, coupled to oxidation by NR and nitrate, yielded similar kinetics for the two dyes. In both cases, second-order kinetics were not obeyed, and reoxidation of dye-reduced Mo-pterin of NR by nitrate became limiting at low scan rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase.

A partial cDNA clone coding for the haem-binding domain of NADH:nitrate reductase (EC 1.6.6.1) (NR) from the unicellular green alga Chlorella vulgaris has been isolated, sequenced and expressed. A 1.2 kb cDNA (pCVNR1) was isolated from a lambda gt11 expression library produced from polyadenylated RNA extracted from nitrate-grown Chlorella cells. pCVNR1 hybridized to a 3.5 kb mRNA transcript that was nitrate-inducible and absent from ammonium-grown cells. The entire sequence of pCVNR1 was obtained and found to have a single uninterrupted reading frame. The derived amino acid sequence of 318 amino acids has a 45-50% similarity to higher-plant NRs, including Arabidopsis thaliana, spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum). A comparison with the putative domain structure of higher-plant nitrate reductases suggested that this sequence contains the complete haem-binding domain, approximately one-third of the Mo-pterin domain and no FAD-binding domain. A 32% sequence similarity is evident when comparing the Chlorella NR haem domain with that of calf cytochrome b5. Expression of pCVNR1 in a pET vector synthesized a 35 kDa protein that was antigenic to anti-(Chlorella NR) antibody. The spectral properties of this protein (reduced and oxidized) in the 400-600 nm region are identical with those of native Chlorella NR and indicate that haem is associated with the protein.

Nitric oxide. New discoveries, biomedical implications.

Nitric oxide (NO) has been identified as a naturally-occurring metabolite in mammalian systems, formed from the amino acid arginine in response to a variety of physiological stimuli. It appears to function in cell-cell communication and may act either directly or through the stimulation of cyclic GMP synthesis in the regulation of such diverse functions as smooth muscle relaxation, inhibition of platelet aggregation and adhesion, central nervous system activity, and cytostasis. The significant role(s) could have important biomedical implications since perturbations in the biosynthesis, release or actions of NO could lead to hypertension, CNS dysfunction or increased susceptibility to infection. Understanding the enzymology and regulation of this pathway in various tissues may lead to development of pharmacological agents specifically designed for the rational control of nitric oxide formation and action.

Spectroscopic, thermodynamic and kinetic properties of Candida nitratophila nitrate reductase.

Visible spectra of oxidized and reduced Candida nitratophila assimilatory NAD(P)H:nitrate reductase yielded absorbance maxima of 413 nm and 423 nm, and 525 nm and 555 nm respectively, characteristic of a b5-type cytochrome. E.p.r. spectra of the partially reduced enzyme revealed a single Mo(V) species (g1 = 1.9957, g2 = 1.9664 and g3 = 1.9658) exhibiting superhyperfine coupling to a single proton [A(1H)av. = 1.4 mT]. Oxidation-reduction midpoint potentials (E'0) (25 degrees C, pH 7) for the haem and Mo-pterin prosthetic groups were determined by visible and e.p.r. potentiometric titrations and yielded values of E'0 = -174 mV (n = 1) for the haem and E'0 = -3 mV and E'0 = -27 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples respectively. Comparison of initial rates of the NADH-oxidizing and nitrate-reducing partial activities at various ionic strengths indicated electron transfer from reduced haem to Mo was rate-limiting during turnover. These results suggest a close similarity between Candida nitratophila and Chlorella vulgaris nitrate reductases.

Oxidation-reduction potentials of flavin and Mo-pterin centers in assimilatory nitrate reductase: variation with pH.

Potentiometric titrations of assimilatory nitrate reductase from Chlorella vulgaris were performed within the pH range 6.0-9.0. Mo(V) was measured by room temperature EPR spectroscopy while the reduction state of FAD was monitored by CD spectroscopy. Between pH 6 and 8.5, the line shape of the Mo(V) EPR signal was constant, exhibiting superhyperfine coupling to a single, exchangeable proton. Potentiometric titrations indicated the Em values for the Mo(VI)/Mo(V) (+61 mV, pH 6) and Mo(V)/Mo(IV) (+35 mV, pH 6) couples decreased with increasing pH by approximately -59 mV/pH unit, consistent with the uptake of a single proton upon reduction of Mo(VI) to Mo(V) and Mo(V) to Mo(IV). The pKa values for the dissociation of these redox-coupled protons appeared to lie outside the pH range studied: pKo(MoVI), pKo(MoV) less than 5.5; pKr(MoV), pKr(MoIV) greater than 9. The Em (n = 2) for FAD (-250 mV, pH 7) varied by approximately -30 mV/pH unit within the pH range 6.0-9.0. Low-temperature EPR potentiometry at the extreme pH values indicated less than 0.5% conversion of FAD to the semiquinone form at the midpoint of the titrations. In contrast, NADH-reduced enzyme exhibited approximately 3-5% of the FAD in the semiquinone form, present as the anionic (FAD.-) species, the spectrum characterized by a line width of 1.3 mT at both pH 6.0 and 9.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation--reduction midpoint potentials of the flavin, haem and Mo-pterin centres in spinach (Spinacia oleracea L.) nitrate reductase.

Oxidation-reduction midpoint potentials have been determined for the flavin, cytochrome b557 and Mo-pterin prosthetic groups of spinach (Spinacia oleracea L.) assimilatory nitrate reductase using visible, c.d. and room-temperature e.p.r. potentiometric titrations. At pH 7 and 25 degrees C, the midpoint potential for the FAD/FADH2 couple was determined by c.d. potentiometry to be -280 +/- 10 mV (n = 2). The redox potential for reduction of the haem was determined by visible potentiometry to be -123 +/- 10 mV (n = 1), significantly lower than the previously published value of -60 mV [Fido, Hewitt, Notton, Jones & Nasrulhaq-Boyce (1979) FEBS Lett. 99, 180-182]. Potentials for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) redox couples, determined by room-temperature e.p.r. potentiometry, were found to be +2 +/- 20 and -6 +/- 20 mV respectively. These values are very similar to the values previously determined for the FAD, haem and Mo-pterin centres in assimilatory nitrate reductase isolated from the unicellular green alga Chlorella vulgaris and indicate a close thermodynamic similarity between the two enzymes.

Chloride inhibition of spinach nitrate reductase.

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V(max) and K(m) for NO(3) (-) yielding V(max) values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K(m) values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K(m) of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K(i) of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO(3) (-) complex. Binding of Cl(-) to the enzyme-NO(3) (-) complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.

Regulation of Chlorella nitrate reductase: control of enzyme activity and immunoreactive protein levels by ammonia.

Nitrate reductase catalyzes the initial step in the conversion of nitrate to organic nitrogen and is thought to be repressed by ammonia and induced by nitrate. Induction by nitrate and repression by ammonia were studied by following changes in NADH:nitrate reductase and the associated partial activities NADH:cytochrome c reductase and methylviologenr:nitrate reductase. Immunoreactive protein was assessed by enzyme-linked immunosorbent assay and immunoblotting. Molybdenum cofactor levels were investigated using the nit-1 complementation assay as well as fluorescence of the oxidized cofactor. The results indicate that the NADH:cytochrome c reductase activity is "induced" faster than the nitrate-reducing activity and suggest that incorporation of the molybdo-pterin cofactor may be rate limiting in the expression of activity. Molybdenum cofactor levels are significantly elevated in nitrate-treated cells. Under "repressing" conditions all activities decreased at approximately the same rate. A more rapid conversion of the enzyme to a reversibly inactive form also occurred under these conditions. Changes in immunoreactive protein levels correlated most closely with NADH:cytochrome c reductase activity but appeared to increase faster during induction and decrease slightly slower during repression than the enzyme activities. Removal of exogenous ammonia results in the appearance of nitrate reducing activity, as well as immunoreactive protein (derepression). Studies using protein and RNA synthesis inhibitors indicated that de novo synthesis is required for nitrate reductase induction and were in agreement with the results of the immunoreactive studies.

Circular dichroism and potentiometry of FAD, heme and Mo-pterin prosthetic groups of assimilatory nitrate reductase.

Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)

Stoichiometry of electron uptake and oxidation-reduction midpoint potentials of NADH:nitrate reductase.

Microcoulometric titrations of NADH:nitrate reductase at 25 degrees C in Mops buffer, pH 7.0, showed that the native enzyme, containing functional FAD, haem and Mo, required addition of five electrons for complete reduction. Reduction of the native enzyme occurred in three waves corresponding to addition of reducing equivalents to the centres in the order: Mo, haem, FAD. Oxidation-reduction midpoint potentials (E'0) for the various redox couples were calculated to be as follows: MoVI/MoV, +16 mV; MoV/MoIV, -27 mV; haemoxidized/haemreduced, -172 mV; FAD/FADH2, -283 mV. The values for the haem and flavin are in excellent agreement with those obtained by visible titrations, namely -164 mV and -288 mV respectively. In contrast, the results for the Mo centre are 28-50 mV more positive than the values previously determined by e.p.r. analysis of frozen enzyme samples poised at defined potentials at 25 degrees C and suggest different pH-dependencies or entropies of reduction for the Mo couples.

Characterization of Nitrate Reductases from Corn Leaves (Zea mays cv W64AxW182E) and Chlorella vulgaris: Sensitivity to a Proteinase Extracted from Corn Roots.

The sensitivity of the two forms of nitrate reductase, NR(I) and NR(II), obtained from the primary leaf of corn, to a limited action corn root proteinase has been examined. The corn inactivating protein (CIP) inhibited the overall reaction (NADH-NR) and the two partial reactions, cytochrome c reductase and reduced methyl viologen NR (MV-NR) of both forms of NR. NADH-cytochrome c reductase was more sensitive to the protease than MV-NR. NR(II) was less sensitive to inactivation than NR(I). When NR(I) and NR(II) were inactivated and then subjected to native gel electrophoresis the protein bands associated with MV-NR activity shifted from an R(m) value of 0.32 to 0.61 for NR(I) and from an R(m) of 0.28 to 0.60 for NR(II). For Chlorella NR these values are 0.32 and 0.70. The initial cleavage of the 116 kilodalton subunit of NR(I) yielded fragments of 84 and 80 kilodaltons after a 5 minute incubation with CIP. With longer incubation times smaller fragments were also identified. For the Chlorella NR the initial cleavage products are approximately 68 and 25 kilodaltons. Longer incubation times also led to smaller fragments. The products of hydrolysis by this limited action protease are quite different for the corn and Chlorella NRs.

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes.

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.