High-resolution (1)H NMR and magic angle spinning NMR spectroscopic investigation of the biochemical effects of 2-bromoethanamine in intact renal and hepatic tissue.

The metabolic consequences of xenobiotic-induced toxicity were investigated using high-resolution magic angle spinning (MAS) NMR spectroscopy of intact tissue. Renal papillary necrosis (RPN) was induced in Sprague-Dawley rats (n = 12) via a single i.p. dose of 250 mg/kg 2-bromoethanamine (BEA) hydrobromide. At 2, 4, 6, and 24 h after treatment with BEA, three animals were killed and tissue samples were obtained from liver, renal cortex, and renal medulla. Tissue samples were also removed at 2 and 24 h from matched controls (n = 6). (1)H MAS NMR spectroscopic techniques were used to analyze samples of intact tissue ( approximately 10 mg). Decreased levels of nonperturbing renal osmolytes (glycerophosphocholine, betaine, and myo-inositol) were observed in the renal papilla of BEA-treated animals at 6 and 24 h postdose (p.d.), concomitant with a relative increase in the tissue concentration of creatine. Increased levels of glutaric acid were found in all tissues studied in BEA-treated animals at 4 and 6 h p.d., indicating the inhibition of mitochondrial fatty acyl CoA dehydrogenases and mitochondrial dysfunction. Increased levels of trimethylamine-N-oxide occurred in the renal cortex at 6 h p.d. Changes in the metabolite profile of liver included an increase in the relative concentrations of triglycerides, lysine, and leucine. The novel application of (1)H MAS NMR to the biochemical analysis of intact tissues following a toxic insult highlights the potential of this technique as a toxicological probe in providing a direct link between urinary biomarkers of toxicity and histopathological evaluation of toxicological lesions.

NMR spectroscopy based metabonomic studies on the comparative biochemistry of the kidney and urine of the bank vole (Clethrionomys glareolus), wood mouse (Apodemus sylvaticus), white toothed shrew (Crocidura suaveolens) and the laboratory rat.

The metabolic profiles of three wild mammals that vary in their trophic strategies, the herbivorous bank vole (Clethrionomys glareolus), the granivorous wood mouse (Apodemus sylvaticus), and the insectivorous white-toothed shrew (Crocidura suaveolens), were compared with that of a widely used strain of laboratory rat (Sprague Dawley). In conjunction with NMR spectroscopic investigations into the urine and blood plasma composition for these mammals, high resolution magic angle spinning (HRMAS) 1H-nuclear magnetic resonance (NMR) spectroscopy was applied to investigate the composition of intact kidney samples. Adaptation to natural diet affects both renal metabolism and urinary profiles, and while these techniques have been used to study the metabolism of the laboratory rat little is known about wild small mammals. The species were readily separated by their urinary profiles using either crude metabolite ratios or statistical pattern recognition. Bank vole urine contained higher concentrations of aromatic amino acids compared with the other small mammals, while the laboratory rats produced relatively more hippurate. HRMAS 1H-NMR demonstrated striking differences in both lipid concentration and composition between the wild mammals and Sprague Dawley rats. Bank voles contained high concentrations of the aromatic amino acids phenylalanine, tyrosine and tryptophan in all tissue and biofluids studied. This study demonstrates the analytical power of combined NMR techniques for the study of inter-species metabolism and further demonstrates that metabolic data acquired on laboratory animals cannot be extended to wild species.

High-resolution (1)H and (1)H-(13)C magic angle spinning NMR spectroscopy of rat liver.

High-resolution magic angle spinning (MAS) (1)H NMR spectra of small samples (ca. 8 mg) of intact rat liver are reported for the first time. One dimensional spectra reveal a number of large well-resolved NMR signals mainly from low to medium molecular weight compounds (generally <1000 Daltons) from a variety of chemical classes. A range of 2D MAS-NMR experiments were performed, including (1)H J-resolved (JRES), (1)H-(1)H total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) to enable detailed signal assignment. Resonances were assigned from alpha- and beta-glucose, glycerol, alanine, glutamate, glycine, dimethylglycine, lysine, and threonine, together with phosphocholine, choline, lactate, trimethylamine-N-oxide (TMAO), and certain fatty acids. Well-resolved (1)H NMR signals from glycogen (poly 1-4 alpha-glucose) were observed directly in intact liver using MAS-NMR spectroscopy. In addition, the resonances from the glycogen C(1)H proton in alpha(1-->4) linked glucose units with either alpha(1-->4) units adjacent or alpha(1-->6) linked branches could be resolved in a high-resolution (1)H NMR experiment giving direct in situ information on the ratio of alpha(1-->4) to alpha(1-->6) units. This indicates that despite the relatively high MW (>1,000,000 Daltons) there is considerable segmental motion in the glycogen molecules giving long (1)H T(2) relaxation times. Magn Reson Med 44:201-207, 2000.

Isoform-specific differences between the type Ialpha and IIalpha cyclic AMP-dependent protein kinase anchoring domains revealed by solution NMR.

Cyclic AMP dependent protein kinase (PKA) is controlled, in part, by the subcellular localization of the enzyme (). Discovery of dual specificity anchoring proteins (d-AKAPs) indicates that not only is the type II, but also the type I, enzyme localized (). It appears that the type I enzyme is localized in a novel, dynamic fashion as opposed to the apparent static localization of the type II enzyme. Recently, the structure of the dimerization/docking (D/D) domain from the type II enzyme was solved (). This work revealed an X-type four-helix bundle motif with a hydrophobic patch that modulates AKAP interactions. To understand the dynamic versus static localization of PKA, multidimensional NMR techniques were used to investigate the structural features of the type I D/D domain. Our results indicate a conserved helix-turn-helix motif in the type I and type II D/D domains. However, important differences between the two domains are evident in the extreme NH(2) terminus: this region is extended in the type II domain, whereas it is helical in the type I protein. The NH(2)-terminal residues in RIIalpha contain determinants for anchoring, and the orientation and packing of this helical element in the RIalpha structure may have profound consequences in the recognition surface presented to the AKAPs.

High-resolution magic angle spinning (1)H NMR spectroscopy of intact liver and kidney: optimization of sample preparation procedures and biochemical stability of tissue during spectral acquisition.

High-resolution magic angle spinning (MAS) (1)H NMR spectroscopy has been used to investigate the biochemical composition of whole rat renal cortex and liver tissue samples. The effects of a number of sample preparation procedures and experimental variables have been investigated systematically in order to optimize spectral quality and maximize information recovery. These variables include the effects of changing the sample volume in the MAS rotor, snap-freezing the samples, and the effect of organ perfusion with deuterated saline solution prior to MAS NMR analysis. Also, the overall biochemical stability of liver and kidney tissue MAS NMR spectra was investigated under different temperature conditions. We demonstrate improved resolution and line shape of MAS NMR spectra obtained from small spherical tissue volume (12 microl) rotor inserts compared to 65 microl cylindrical samples directly inserted into the MAS rotors. D(2)O saline perfusion of the in situ afferent vascular tree of the tissue immediately postmortem also improves line shape in MAS NMR spectra. Snap-freezing resulted in increased signal intensities from alpha-amino acids (e.g., valine) in tissue together with decreases in renal osmolytes, such as myo-inositol. A decrease in triglyceride levels was observed in renal cortex following stasis on ice and in the MAS rotor (303 K for 4 h). This work indicates that different tissues have differential metabolic stabilities in (1)H MAS NMR experiments and that careful attention to sample preparation is required to minimize artifacts and maintain spectral quality.

Group discussion as interactive dialogue or as serial monologue: the influence of group size.

Current models draw a broad distinction between communication as dialogue and communication as monologue. The two kinds of models have different implications for who influences whom in a group discussion. If the discussion is like interactive dialogue, group members should be influenced most by those with whom they interact in the discussion; if it is like serial monologue, they should be influenced most by the dominant speaker. The experiments reported here show that in small, 5-person groups, the communication is like dialogue and members are influenced most by those with whom they interact in the discussion. However, in large, 10-person groups, the communication is like monologue and members are influenced most by the dominant speaker. The difference in mode of communication is explained in terms of how speakers in the two sizes of groups design their utterances for different audiences.

In and on: investigating the functional geometry of spatial prepositions.

Spatial prepositions such as in and on seem to denote semantically indeterminate spatial relations. This reflects, in part, the physical relationships between the objects in the scenes that they are used to portray. We argue that such physical relationships are best represented in terms of an inherently dynamic functional geometry which incorporates notions of location control. Two experiments are reported. They investigate the degree to which independent judgements of location control predict choice of description across a range of scenes. The results show that judgements of location control predict viewer's choice of description under certain circumstances. In the absence of prototypical geometric relations, control information has a strong influence on choice of description. However, when the scenes portray prototypical geometric relations, control information has less of an effect. The results support a hybrid account of the semantic representation underlying the prepositions with both a geometric and a functional component to it.

High-resolution magic angle spinning 1H NMR spectroscopic studies on intact rat renal cortex and medulla.

High-resolution magic angle spinning 1H NMR (MAS-NMR) spectroscopy was used to investigate the biochemical composition of normal renal cortex and renal papilla samples from rats, and results were compared with those from conventional 1H NMR analysis of protein-free tissue extracts. 1H MAS NMR spectra of samples obtained from inner and outer cortex were found to be broadly similar in terms of metabolite profile, and intra- and inter-animal variability was small. However, the MAS NMR spectra from renal papilla samples were qualitatively and quantitatively different from those obtained from cortex. High levels of free amino acids and several organic acids were detected in the cortex, together with choline, glucose, and trimethylamine-N-oxide. The dominant metabolite resonances observed in papillary tissue were from glycerophosphocholine (GPC), betaine, myo-inositol, and sorbitol. On increasing the magic angle spinning rate from 4,200 to 12,000 Hz, the lipid MAS 1H NMR signal profile remained largely unchanged in papillary tissue, whereas "new" resonances from triglycerides appeared in the spectra of cortical tissue, this effect being reversible on returning the spinning rate to 4,200 Hz. Further investigation into the behavior of the lipid components under different spinning rates suggested that the lipids in the cortex were present in more motionally constrained environments than those in the papilla. 1H MAS NMR spectra of tissues are of value both in interrogation of the biochemical composition of whole tissue, and in obtaining information on the mobility and compartmentalization of certain metabolites.

How groups co-ordinate their concepts and terminology: implications for medical informatics.

Conceptual and terminological systems are established and maintained by the communities who use them. This paper reports experiments which investigate the role of communication and interaction in the process. The experiments show that isolated pairs of communicators and virtual communities of interacting pairs naturally converge on their own conceptual and terminological systems when confronted with a common task. The results also indicate that the system converged on is optimal for that particular group engaged in that particular task. These findings are discussed in relation to the increasing use of tightly coordinated medical teams and its implications for getting them to adopt standardized medical terminologies.

Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations.

To develop an alternative approach to measure peptidyl backbone flexibility and to expand our understanding of the segmental flexibility of cAMP-dependent protein kinase (cAPK), the effect of protein kinase inhibitor peptide, PKIalpha(5-24), and MgATP on the mobility of fluorescein selectively conjugated to five sites on the catalytic subunit of cAPK was examined. Specifically, five full-length, single-site catalytic subunit mutants (K16C, K81C, I244C, C199A, and N326C) were prepared, and fluorescein maleimide was selectively attached to the side chains of each substituted cysteine or, in the case of the C199A mutant, to the unprotected native C343. The time-resolved anisotropy decay profiles of the five fluorescein maleimide-conjugated mutants were well fit to a biexponential equation. The fast rotational correlation times of the fluorescein conjugates ranged between 1.9 and 2.8 ns and were inversely correlated (r = -0.87) to the averaged crystallographic main-chain atom B factors around each site of conjugation. The slow correlation times ranged between 25 and 28 ns and were about the same magnitude as the value of 21 ns estimated from the Stokes-Einstein equation. The presence of MgATP and PKIalpha(5-24), which induces the closed conformation of cAPK, was associated with a reduction of the fast rotational correlation time of the K81C conjugate, indicating that the peptidyl backbone around K81 is measurably less flexible when the C subunit is in the closed compared with the open conformation. The results suggest (i) that time-resolved fluorescence anisotropy can assess the nanosecond flexibility of short segments of the peptidyl backbone around each site of labeling and (ii) that the substrate/pseudosubstrate binding differentially affects the backbone flexibility of cAPK.

Synergistic binding of nucleotides and inhibitors to cAMP-dependent protein kinase examined by acrylodan fluorescence spectroscopy.

We have engineered an acrylodan-modified derivative of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) whose fluorescence emission signal has allowed the synergistic binding between nucleotides and physiological inhibitors of cAPK to be examined (Whitehouse, S., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3682-3692). In the presence of the regulatory subunit, RI, the affinity of cAPK for adenosine, ADP, AMPPNP (adenosine 5'-(beta, gamma-imino)triphosphate), or ATP was 5-, 50-, 120-, and 15,000-fold enhanced, while in the presence of the heat-stable inhibitor protein of cAPK (PKI), there was a 3-, 20-, 33-, and 2000-fold enhancement in the binding of these nucleotides, respectively. A short inhibitor peptide, PKI-(14-22), enhanced the binding of ADP to the same degree as did full-length PKI (20-fold) but, in contrast, did not significantly enhance the binding of ATP or AMPPNP. The full binding synergism between PKI and either ATP (2000-fold) or AMPPNP (33-fold) to cAPK could, however, be mimicked by a longer peptide, PKI-(5-24), suggesting that the PKI NH2 terminus (residues 5-13) is most likely critical. Since this region is remote from the ATP gamma-phosphate, the binding synergism must arise through an extended network communication mechanism between the PKI NH2 terminus and the ATP binding site.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

Conversation, co-ordination and convention: an empirical investigation of how groups establish linguistic conventions.

Two experiments are reported which demonstrate the development of co-ordinated description languages in two groups of communicators playing Garrod and Anderson's (1987) maze game. The experiments contrast language co-ordination between speakers who always interact with the same partner (isolated pairs) as compared with speakers who interact with different partners drawn from the same community. Whereas the isolated pairs show higher degrees of inter-speaker convergence than the community pairs at the start of the experiment, the situation reverses by the time they have all played six or more games. The results are discussed at two levels: (1) in terms of Lewis's formal theory of conventions, and (2) in relation to a language processing model which abides by the "output/input co-ordination" principle proposed in Garrod and Anderson (1987).

Evidence indicating proximity in the nucleosome between the histone H4 N termini and the globular domain of histone H1.

Proteolysis of rat liver chromatin by the Arg-C peptidase, clostripain, is characterized by a progressive fragmentation of the N-terminal segments of the four core histones H2A, H2B, H3 and H4, until a well-defined limit digest is reached. This work addresses the case of histone H4. Two intermediate proteolytic sites are identified for this histone, i.e. Arg3 and Arg17, before the limit digest is achieved through cleavage of the polypeptide chain after Arg19. The accessibility of these intermediate sites depends strongly on the presence or absence of histone H1. When H1 is absent, both intermediate sites of histone H4 are similarly accessible, whereas one of them, Arg3, becomes totally inaccessible in the presence of histone H1. Di- and trinucleosomes were used with the aim of avoiding any interference with superstructural effects which can occur with longer polynucleosomes in the presence of H1. We also investigated the accessibility of the Arg sites of H1 that are located primarily in the central globular domain of this histone. In free histone H1, all the centrally located Arg sites are accessible to clostripain. In contrast, in the chromatin-bound state none of these sites is accessible. Besides the arginyl sites in the central globular domain of H1, two Arg residues are observed with the most abundant H1d variant in rat chromatin, one in the N-terminal region and the other in the C-terminal region. The restricted number of proteolytic fragments observed with chromatin-bound H1 is accounted for by the cleavage of H1 after these Arg residues located on the outside of the globular domain. Our results suggest that mutual steric effects are at play between histones H1 and H4 and indicate that the N termini of both histones H4 in the nucleosome lie in close proximity to the globular domain of H1. Based on these observations and taking into account the known structural features of the nucleosome, we propose a model for positioning the N-terminal segments of both histones H4 at the periphery of the nucleoprotein structure. In this model both H4 segments are located within the expanded DNA minor grooves, at periods +/- 1, symmetrically disposed relatively to the nucleosome dyad axis. This arrangement brings the amino ends of both H4 molecules in close contact with the H1 globular domain thus accounting for the observed inaccessibility of the Arg3 site of H4 in the presence of H1.

Probing the specificity of nucleotide binding to the F1-ATPase from thermophilic Bacillus PS3 and its isolated alpha and beta subunits with 2-N3-[beta, gamma-32P]ATP.

Irradiation of the F1-ATPase from Bacillus PS3 (TF1) in the presence of 134 microM 2-N3-[beta, gamma-32P]ATP plus Mg2+ for 90 min led to 95% inactivation of the ATPase activity which was accompanied by exclusive labeling of the beta subunit. The isolated alpha and beta subunits were also treated separately with 2-N3-[beta, gamma-32P]ATP under similar conditions. Fractionation of a tryptic digest of photolabeled TF1 by reversed-phase HPLC resolved a major and a minor radioactive peptide. Sequence analyses showed that the major peptide contained labeled Tyr-beta 364, whereas the minor one contained labeled Tyr-beta 341, residues known to be part of noncatalytic and catalytic sites, respectively. Two closely eluting radioactive peptides were obtained when a tryptic digest of the photolabeled, isolated beta subunit was fractionated by HPLC. Sequence analyses revealed that both contained labeled Tyr-beta 341. Fractionation of a tryptic digest of the photolabeled, isolated alpha subunit by HPLC resolved two peptides which contained the majority of the radioactivity incorporated. When subjected to eight cycles of automatic Edman degradation, one gave the sequence APGVXDR, corresponding to residues 133-139, in which X is a gap and corresponds to Met-alpha 137, which presumably is the derivatized residue. Only the first five cycles yielded phenylthiohydantoin derivatives when the other radioactive peptide derived from the alpha subunit was submitted to automatic Edman degradation which revealed the sequence APGVM, suggesting that Asp-alpha 138 is derivatized. The overall results suggest that the isolated beta subunit is a useful model for studying binding of nucleotides to catalytic sites, whereas the isolated alpha subunit may be of limited value in modeling interactions of nucleotides with noncatalytic sites.

Ultraviolet transmittance of contact lenses.

Chronic and acute exposure to ultraviolet (UV) radiation has been shown to have deleterious effects on all layers of the human eye. As a consequence, appropriate protective measures have been advocated with respect to exposure to this form of radiation. Among suggested actions are spending less time in the sun and wearing protective visual aids such as those containing UV-absorbing materials when exposed to sunlight. To investigate the protective ability of currently available contact lens materials we measured the transmittance characteristics of 11 different types of soft and rigid contact lenses in the UV and visible range from 280 to 500 nm with a Zeiss model DM4 Dual Beam Spectrophotometer. Our findings indicate that lenses not treated with UV absorbers transmitted most of the UV radiation. Lenses containing UV absorbers provided excellent UV protection and transmitted significantly less UV radiation than the untreated lenses (Student's t-test with Bonferoni's correction, N = 10, p < 0.001). Based on our findings, we recommend that contact lenses with UV absorbers be considered as a viable option for providing UV protection, especially for aphakic patients, patients taking photosensitizing pharmaceutical agents, and those patients who spend a great deal of time outdoors.

Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase expressed in Escherichia coli is a phosphoprotein. By in vivo labeling with [32Pi]orthophosphate, the sites of phosphorylation were identified as Ser-10, Ser-139, Thr-197, and Ser-338. Two of these sites, Thr-197 and Ser-338, are found in the mammalian enzyme (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). The predominant isoform is phosphorylated at Ser-10, Ser-338, and Thr-197. The isoforms cannot be readily interconverted by in vitro autophosphorylation, suggesting that the phosphates are relatively stable once the mature protein is assembled. Unlike the mammalian enzyme, the recombinant enzyme is not myristylated at its animo terminus. By coexpressing the catalytic subunit and N-myristyl transferase, the recombinant catalytic subunit is myristylated, and, under these conditions, phosphorylation at Ser-10 is reduced. The fact that recombinant catalytic subunit mutants that are enzymatically impaired are not phosphorylated in vivo indicates that the phosphorylation of the catalytic subunit observed in E. coli is due to autophosphorylation. Whether this process is intramolecular or intermolecular cannot be distinguished. Although autophosphorylation accounts for the modification of the catalytic subunit when it is expressed in E. coli, there may be heterologous protein kinases that are responsible for its in vivo phosphorylation when the enzyme is expressed in eukaryotic cells.

Discourse influences during parsing are delayed.

Subjects read sentences containing either a syntactically ambiguous prepositional phrase attachment or a syntactically ambiguous reduced relative clause. The sentences were embedded in passages of text that were consistent with either the minimal or non-minimal attachment reading. In addition, a discourse factor (i.e., whether or not the target sentence was in the discourse focus) was varied. Subjects' eye movements were recorded as they read the passages of text. Our primary finding was that subjects were garden-pathed even when there was biasing context. However, when the target sentence was in the discourse focus, subjects were able to recover more readily from their initial erroneous parse of the sentence. The data thus support models of sentence parsing that postulate that the parsing of a sentence is based upon structurally based principles and the influence of semantic or pragmatic information makes itself felt only after the initial parsing decision has been made.

The Human Communication Research Centre dialogue database.

The HCRC dialogue database consists of over 700 transcribed and coded dialogues from pairs of speakers aged from seven to fourteen. The speakers are recorded while tackling co-operative problem-solving tasks and the same pairs of speakers are recorded over two years tackling 10 different versions of our two tasks. In addition there are over 200 dialogues recorded between pairs of undergraduate speakers engaged on versions of the same tasks. Access to the database, and to its accompanying custom-built search software, is available electronically over the JANET system by contacting liz@psy.glasgow.ac.uk, from whom further information about the database and a user's guide to the database can be obtained.

Irradiation of the bovine mitochondrial F1-ATPase previously inactivated with 5'-p-fluorosulfonylbenzoyl-8-azido-[3H]adenosine cross-links His-beta 427 to Tyr-beta 345 within the same beta subunit.

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p'-fluorosulfonylbenzoyl-8-azidoadenosine (8-N3-FSBA) with an apparent Kd of 0.47 mM at pH 8.0 and 23 degrees C in the absence of light. Irradiation of dark-inactivated enzyme with long-wavelength UV light produced cross-linked dimers and, to a lesser extent, trimers made up of alpha and beta subunits. Two major radioactive peptides were resolved by high-performance liquid chromatography from tryptic digests of MF1 which had been inactivated with 8-N3-FSB[3H]A at pH 8.0 in the dark. Sequence analysis revealed that one contained Tyr-beta 368 and the other contained His-beta 427 which were labeled in the ratio of 18:15. Sequence analysis of radioactive tryptic peptides isolated from digests of irradiated MF1 derivatized with 8-N3-FSB[3H]A showed that photolysis induced cross-linking of His-427 to Tyr-345 within the same beta subunit in high yield. When MF1 derivatized with 8-N3-FSB[3H]A was irradiated in the presence of beta-mercaptoethanol, alpha-beta cross-links were eliminated, whereas those between His-beta 427 and Tyr-beta 345 were unaffected. Analysis of radioactive peptides in tryptic digests of MF1 derivatized with 8-N3-FSB[3H]A and then irradiated in the presence or absence of beta-mercaptoethanol showed that the nitrene generated from reagent attached to Tyr-beta 368 participates in formation of alpha-beta cross-links in the absence of beta-mercaptoethanol. Therefore, the nitrene generated from reagent tethered to His-beta 427 is shielded from solvent and reacts with the side chain of Tyr-beta 345. In contrast, the nitrene generated from reagent attached to Tyr-beta 368 is exposed to solvent, but in the absence of scavengers reacts with side chains present in the alpha subunit. Irradiation of MF1, partially inactivated with 8-N3-FSBA, led to loss of residual ATPase activity without affecting residual ITPase activity. The amount of photoinactivation was greater when partial dark inactivation was performed at pH 6.9, where modification of His-beta 427 predominates, than when performed at pH 8.0, where modification of Tyr-beta 368 predominates. This suggests that cross-linking of His-beta 427 to Tyr-beta 345, and not cross-linking of alpha and beta subunits, is responsible for the augmented inactivation induced by irradiation.