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Two patients had a previously unrecognized form of crystallocryoglobulinemia. Their clinical presentations were similar, consisting of necrotizing vasculitis and purpura involving the legs. Analysis of each cryoglobulin complex showed that two components, albumin and a monoclonal IgG-lambda, were present, and both components were needed in a fixed ratio for precipitation. In addition, cryoprecipitation occurred in serum, but not plasma, due to citrate inhibition of complex formation. Our findings suggest that the monoclonal IgGs have the properties of antibodies directed specifically against a calcium-dependent antigenic site on human albumin, and that the resultant IgG-lambda-albumin immune complexes crystallized in the cold. [ABSTRACT FROM AUTHOR]

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted alpha-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 micrograms/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.

The retroviral nucleocapsid protein (NC) associates, histone-like, with genomic RNA within the viral capsid. NC, an essential component of replication competent retroviruses, is also associated with events leading both to virus assembly and to reverse transcription. The nucleic acid binding properties of NC are key to understanding these properties, yet only a minimal biochemical description of NC-nucleic acid interactions is available. We have used the anisotropy of the intrinsic fluorescence of NC from avian myeloblastosis virus to quantify its binding to a variety of nucleic acids. Using salt back-titrations, the intrinsic equilibrium association constant per nucleic acid site, K(obs), was determined for NC binding to single- and double-stranded RNAs and DNAs. In 0.125 M NaCl, 40 mM HEPES at pH 7.0 and 27 degrees C, the log K(obs) ranged from 3.3 to 4.0 (average 3.7) for these nucleic acids. From the salt dependence of K(obs), it was estimated that, on balance, 1 ion was displaced upon formation of each complex; it is likely that cation displacement from nucleic acid is offset by anion binding by protein during complex formation. The logarithm of the mean intrinsic affinity in the absence of polyelectrolyte effects, log KT, was 3.1, corresponding to a delta G of -4.2 kcal/mol. K(obs), KT, and the number of displaced ions were independent of pH between pH 5.6 and 8.9, indicating that NC residues that titrate in this pH range are not contributing to binding. K(obs) and KT increase with temperature, in the range 15 to 47 degrees C. From van't Hoff analysis, entropy was found to be the driving force for formation of the NC-poly(rA) complex, even in the absence of the polyelectrolyte effect. The general nature of NC interactions with nucleic acids is shown by the similarity of the K(obs) values for RNAs and DNAs in both single-stranded and double-stranded structures. This ability of NC to interact with all types of nucleic acids may provide it with the necessary versatility to function like a histone in facilitating the packaging of viral RNA and yet function early in infection, where it has been ascribed a role in facilitating reverse transcription.

We have exploited the sole tryptophan residue (Trp535) in the ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) to study features of the isolated polypeptide (p15 RNase H) by fluorescence spectroscopy. Incubation of purified p15 RNase H with a synthetic RNA/DNA hybrid was accompanied by an alteration in Trp535 fluorescence intensity. This property was used to determine an apparent binding constant (Kapp) of 3.5 x 10(6) M-1 for p15 RNase H complexed with poly(rA)/oligo(dT)12-18 and an occluded site size of 4 nucleotides. A cooperativity coefficient (omega) of 910 was also determined which indicated that nearly three logs of the Kapp were due to cooperativity effects. Recombinant p15 RNase H preparations containing mutations at position 478 (Glu478-->Gln478) or 539 (His539 -->Phe539), which are highly conserved between bacterial and retroviral RNases H, were also analyzed. Under the same conditions, these mutants failed to bind the RNA/DNA hybrid, although they were structurally similar to the wild type polypeptide. Fluorescence spectroscopy thus appears to be an alternative and sensitive means of analyzing functional properties of the purified RNase H domain of HIV-1 RT under a variety of conditions.

The glucokinase (EC 2.7.1.63) from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using inorganic polyphosphate (poly(P)) or ATP as the phosphoryl donor. The nature of the poly(P) and ATP sites was investigated by using N-bromosuccinimide (NBS) as a probe for the involvement of tryptophan in substrate binding and/or catalysis. NBS oxidation of the tryptophan(s) resulted in fluorescence quenching with concomitant loss of both the poly(P)- and ATP-dependent glucokinase activities. The inactivation by NBS was not due to extensive structural changes, as evidenced by similar circular dichroism spectra and fluorescence emission maxima for the native and NBS-inactivated enzyme. Both phosphoryl donor substrates in the presence of xylose afforded approximately 65% protection against inactivation by NBS. The Km values of poly(P) and ATP were not altered due to the modification by NBS, while the catalytic efficiency of the enzyme was decreased, suggesting that the essential tryptophan(s) are involved in the catalysis of the substrates. Acrylamide quenching studies indicated that the tryptophan residue(s) were partially shielded by the substrates against quenching. The Stern-Volmer quenching constant (KSV) of the tryptophans in unliganded glucokinase was 3.55 M-1, while KSV values of 2.48 and 2.57 M-1 were obtained in the presence of xylose+poly(P)5 and xylose+ATP, respectively. When the tryptophan-containing peptides were analyzed by peptide mapping, the same peptide was found to be protected by xylose+poly(P)5 and xylose+ATP against oxidation by NBS. The two protected peptides were determined to be identical by N-terminal sequence analysis and amino acid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-derived growth factor-AA and -BB homodimers and -AB heterodimers bind with high affinity to the platelet-derived growth factor (PDGF) alpha-receptor. Basic polypeptides such as polylysine and protamine sulfate compete with PDGF for receptor binding, suggesting a role for ligand positive charge in the binding interaction. A pentapeptide amino acid sequence with a cationic tripeptide core is perfectly conserved between the A- and B-chains (Val158-Arg159-Lys160-Lys161-Pro162) and was therefore considered as a possible alpha-receptor-binding domain. We have investigated the functional importance of positive charge within this region of the PDGF A-chain by using site-directed mutagenesis to convert the cationic core amino acids to the acidic sequence triglutamic acid. cDNAs encoding wild-type (PDGF-AAwt) and charge mutant (PDGF-AAcm) proteins were expressed following stable transfection of Chinese hamster ovary cells. Proper assembly and secretion of PDGF-AAcm was verified by metabolic labeling with [35S]cysteine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis analysis under nonreducing and reducing conditions. PDGF-AAcm was secreted as two major species of disulfide-linked A-chain homodimers identical in molecular mass to those observed for PDGF-AAwt (32 and 35 kDa). Secreted PDGF-AAwt and PDGF-AAcm proteins were purified to homogeneity and subjected to structural and functional analyses. Compared to purified PDGF-AAwt, PDGF-AAcm displayed a marked reduction in both binding affinity for PDGF alpha-receptors and mitogenic activity in Swiss 3T3 cells. Large reductions were also observed in the ability of semipurified PDGF-AAcm to stimulate calcium influx and the production of inositol phosphates. Measurement of circular dichroism spectra of highly purified PDGF-AAcm and PDGF-AAwt revealed no significant difference in secondary structure. Collectively, these results indicate that the cationic Arg159-Lys160-Lys161 region plays a critical role in the biological activity of PDGF-AA by direct participation in ligand binding to the PDGF alpha-receptor.

Polyphosphate [poly(P)n]:D-(+)-glucose-6-phosphotransferase (EC 2.7.1.63) from Mycobacterium tuberculosis H37Ra was purified to homogeneity using an improved method which yielded a 634-fold purification with higher recovery. The purified enzyme migrated as a single band with M(r) 33 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native enzyme was shown to be a dimer by gel filtration using high-performance liquid chromatography (HPLC). The purified enzyme fractionated as a single peak on a C8 reverse-phase HPLC column and was found to display both polyphosphate- and ATP-dependent glucokinase activities. Further evidence that a single protein was responsible for both activities was shown by nondenaturing PAGE, in which the two activities (as determined by an activity stain in dual experiments) were found to comigrate. The C-terminal analysis yielded a single sequence while the N-terminus which was blocked also yielded a single sequence after deblocking. The two activities were found to have the same temperature optimum of 50 degrees C. The pH optima were 9.5 and 8.6-9.5 with poly(P)32 and ATP as the phosphoryl donors, respectively. The apparent Km for poly(P)32 was 18.4 microM while the Km for ATP was 1.46 mM. In addition, the nucleotide analogue, Reactive Blue 4, was found to be a competitive inhibitor with ATP in the ATP-dependent glucokinase reaction, while it displayed noncompetitive inhibition patterns with poly(P) in the poly(P)-dependent glucokinase reaction. It is concluded that the poly(P) and ATP glucokinase activities are catalyzed by the same enzyme but that the two substrates may have different binding sites.

The hypothesis that dihydrolipoamide dehydrogenases (E3s) have tertiary structures very similar to that of human glutathione reductase (GR) was tested in detail by three separate criteria: (1) by analyzing each putative secondary structural element for conservation of appropriate polar/nonpolar regions, (2) by detailed comparison of putative active site residues in E3s with their authentic counterparts in human GR, and (3) by comparison of residues at the putative dimeric interface of the E3s with the authentic residues in GR. All three criteria are satisfied in a convincing way for the 7 E3s that were considered, supporting the conclusion that the structural scaffolding and the overall tertiary structure (which determines the location of functional sites and residues) are remarkably similar for the E3s and for GR. These analyses together with the crystal structures of human erythrocyte GR formed the basis for construction of a molecular model for human E3. The cofactor FAD and the substrates NAD and lipoic acid were also included in the model. Unexpectedly, the surface residues in the cleft that holds the lipoamide were found to be highly charged and predominantly acidic, allowing us to predict that the region around the lipoamide in the subunit should be basic in nature. The molecular model can be tested by site-directed mutagenesis of residues predicted to be in the dihydrolipoamide acetyltransferase subunit binding cleft.

The interaction of the retroviral nucleocapsid protein (NC) with nucleic acids forms the basis of its varied roles in the replication cycle, which include binding and condensing the viral RNA within the virion, stimulation of the early steps in reverse transcription, and dissociation from RNA in the replication complex. As part of an investigation of the NC binding site and of the forces that drive its interaction with nucleic acids, the relative affinities of NC from avian myeloblastosis virus were determined for a series of mononucleotides and mononucleotide components using a competitive displacement assay utilizing the extrinsic fluorescent probe bis-ANS [Secnik, J., Wang, Q., Chang, C.-M., & Jentoft, J.E. (1990) Biochemistry 29, 7991-7997]. The estimated binding affinities were unexpectedly similar for nucleotides, nucleosides, and bases (Ka greater than 10(6) M-1). AMP, UMP, GMP, and CMP bound to NC with essentially equal affinity, indicating that NC does not discriminate between bases. This is consistent with its role in coating, condensing, and packaging the RNA within virions. Nucleosides, bases, riboses, and ribose phosphate bind to NC with 1000-fold higher affinity than inorganic phosphate, indicating that the NC binding site includes elements that recognize nucleotide base and ribose components in addition to phosphate ions. However, the binding affinities of components are not additive, i.e., the Kapp values for adenine and deoxyribose are very similar to that for deoxyadenosine, indicating that the interaction between the NC subsite and the base and the sugar components is complex. The stoichiometry of the complex between bis-ANS and NC was established to be NC.(bis-ANS)3.(ABSTRACT TRUNCATED AT 250 WORDS)

Immature female rats (23-30 days old) were implanted subcutaneously with diethylstilbestrol (DES) in silastic capsules. After 48 h their ovaries were removed and the granulosa cells isolated (Foreman et al. (1984) Life Sci. 35, 1273-1279). The cells were incubated in Hepes balanced saline buffer with substrates with or without follicle-stimulating hormone (FSH). At the end of incubation perchloric acid extracts were made for 31P NMR spectroscopy. The resonances of fructose 1-phosphate, fructose 6-phosphate, glucose 1-phosphate, and ribose 5-phosphate were identified in the granulosa cell extracts. The relative intensities of fructose 6-phosphate to ribose 5-phosphate decreased after incubation with FSH in vitro. This suggests that FSH increases the activity of the pentose pathway within 1 h. Thus, FSH can acutely activate those metabolic pathways which provide nicotinamide-adenine dinucleotide phosphate (NADPH) to be used in steroid synthesis and cholesterol mobilization.

The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of bis-ANS is enhanced many fold upon forming a complex with the NC. Between 2 and 10 molecules of bis-ANS bind strongly to the NC, with an overall Kd of less than 10(-6) M. The emission of bis-ANS in the complex can also be induced by excitation at 298 nm, indicating that energy is transferred from Trp 80, the sole tryptophan in the NC protein, to bis-ANS. The energy transferred between the Trp 80 and bis-ANS was analyzed to yield a calculated distance of separation between these fluorophores of 28 +/- 3 A; thus, Trp 80 is well removed from the nearest bound bis-ANS. The fluorescence emission of bis-ANS in the NC.bis-ANS complex is efficiently quenched by added salts and by poly(A), suggesting that salt (presumably anions), nucleic acid, and bis-ANS bind to the same, positively charged region on the NC protein. A site size of six nucleotides was determined for nucleic acid binding to the NC protein, with an estimated Kd of less than 10(-6) M. Salt (anion) binding is strong, but nonspecific, with a Kapp of 4 mM, raising the possibility that anion binding to the NC protein might regulate the interaction of the NC with viral RNA inside the host cell.

An unusual human cryoglobulin complex was characterized as a two-component system containing monoclonal immunoglobulin G (IgG) and serum albumin in a 1:2 mole ratio. This complex appeared to be an antibody-antigen complex, since the mole ratio was appropriate and the isolated Fab of the IgG associated with the albumin. The cryoglobulin apparently arose as a result of specific association and/or aggregation of the IgG albumin adduct, since the cryoglobulin complex formed a crystalline precipitate. The IgG and albumin were separated and characterized with respect to immunological cross-reactivities, sedimentation velocities, isoelectric properties, and amino acid composition. The extent of precipitation of the cryoglobulin complex was maximal at pH 7.8, was decreased by added ions including citrate, ethylenediaminetetraacetic acid, NaCl, and CaCl2, and was decreased by increasing temperature. Both the nature of the cold-precipitable complex and the solubility properties differed from those described for other cryoglobulins.

Yeast hexokinase isozymes P1 and P11 exhibit a pH dependent, rapid relaxation process at 15 degrees C at enzyme concentrations of 100-474 muM and over a pH range of 6-8. The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red. The value of 1/tau varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for P1 and 85 ms-1 for P11 at pH 6. The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization. The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 X 10(11) M-1 S-1; the estimated pKa varies between 5.5 and 6.1. The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes. The isomerization rate is at least 50 times faster than catalysis under all conditions and the pKa is lower than that controlling activity. The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed. These data imply that isomerizing and ionizing forms are sensitive to events at the active site. These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme.

A unique resonance in the 13C NMR spectrum of [13C]methylated ribonuclease A has been assigned to a N epsilon, N-dimethylated active site residue, lysine 41. The chemical shift of this resonance was studied over the pH range 3 to 11, and the titration curve showed two inflection points, at pH 5.7 and 9.0. The higher pKa, designated pKa1, was assigned to the ionization of the lysyl residue itself while the pKa of 5.7, designated pKa2, was assigned on the basis of its pKa to the ionization of a histidyl residue which is somehow coupled to lysine 41. Both pKa values are measurably perturbed by the binding of active site ligands including nucleotides, nucleosides, phosphate, and sulfate. In most cases, the alterations in pKa values induced by the ligands were larger for pKa2. The ligand-induced perturbations in pKa2 generally paralleled those reported for histidine 12, another active site residue (Griffin, J. H., Schechter, A. N., and Cohen, J. S. (1973) Ann. N. Y. Acad. Sci. 222, 693-708). The sensitivity of the N epsilon, N-dimethylated lysine 41 resonance to the histidyl ionization may result from a conformational change in the active site region of ribonuclease which is coupled to the histidyl ionization. This coupling between lysine 41 and another ribonuclease residue, which has not been documented previously, offers new insight into the interrelationship between residues in the active site of this well characterized enzyme.

The availability of the primary amino acid sequences of the E2 of PDC, alpha-KGDC and BCKADC from several prokaryotic and eukaryotic species has allowed us to compare the structural aspects of human PDC-E2 with those of the E2 components from the other complexes. The PDC-E2 components from all the species examined so far contain three structurally identifiable regions: the lipoyl-bearing domain, the E3-binding site, and the catalytic domain. The primary structure of the lipoyl-bearing domain shows considerable variation in its size, ranging from one to three repeating units of approximately 110 amino acids, but essentially preserving its function in the E2 components. In contrast, the sizes of the E3-binding site and the catalytic domain of PDC-E2 from several species are essentially similar and show considerable conservation of specific amino acid residues. Obviously, additional studies are warranted to better understand the structure-function relationships of these domains and the evolutionary conservation of PDC-E2 in different species. Similarly, the availability of the primary amino acid sequences of E3 from several prokaryotes and eukaryotes has also permitted comparison of the structural domains of these proteins with that of the known structure of human GR, a flavoprotein member of the pyridine nucleotide-disulfide oxidoreductase family. Four structural domains (FAD, NAD+, central, and interface domains) have been identified in the E3 components. On the basis of the comparison of the secondary structural elements of GR and E3, the core structure of these two proteins are shown to be similar. It is hoped that further analysis of E3 using site-directed mutagenesis and determination of its crystal structure will provide better insight into its structure-function relationships.

The mode of association of an unusual human autoantibody complex, composed of a monoclonal immunoglobulin, Tu IgG, and human serum albumin was investigated. A crystalline complex forms from these components in the cold and we have shown that it consists of IgG and albumin in a 1:2 molar ratio [Jentoft et al., Biochemistry 21, 289-294 (1982)]. The crystalline complex was analyzed by electron microscopy and the soluble natural complexes (formed by dissolving the crystals at 20 degrees C) were studied by sedimentation velocity. The sedimentation studies demonstrated that the soluble Tu IgG-albumin complexes are in equilibrium with free Tu IgG and albumin molecules and that the major soluble sedimenting species has a S20,w value of 12.5S. At a constant concn of complex, the size of the sedimenting complex can be reduced by lowering the pH, increasing the ionic strength, or adding CaCl2, citrate, ascorbate or urea. These intermediate, soluble forms have S20,w values that are consistent with 1:1 and 1:2 Tu IgG-albumin complexes. Parameters of repeat distances and angles that were obtained from electron micrographs of the crystalline form of the Tu IgG-albumin complex were used to propose a model for the 12.5S species and were also incorporated into a three-dimensional model for the complex. The 12.5S complex is proposed to form by dimerization of the 1:2 Tu IgG-albumin complex via interactions of albumin with the Fc region of the antibody. The 12.5S dimer may be the nucleating species for subsequent rapid associations that lead to spontaneous formation of crystals. In the proposed model for the Tu IgG-albumin crystals, the angle between the Fab arms of each Tu IgG molecule is 90 degrees, the antigenic determinant on the albumin is located near one end of the long axis of the cylindrical molecule, the site of interaction with Fc is located at the other end of the cylinder, and the CH3 domain of the IgG contains the binding site for albumin that is responsible for the formation of the dimeric 12.5S species. A series of sedimentation velocity experiments suggest that the association between the CH3 domain of IgG and albumin requires the prior formation of the antibody-antigen complex.

A BASIC interactive graphics program has been developed for the IBM-PC which utilizes the graphics capabilities of that computer to display and manipulate protein structure from coordinates. Structures may be generated from typed files, or from Brookhaven National Laboratories' Protein Data Bank data tapes. Once displayed, images may be rotated, translated and expanded to any desired size. Figures may be viewed as ball-and-stick or space-filling models. Calculated multiple-point perspective may also be added to the display. Docking manipulations are possible since more than a single figure may be displayed and manipulated simultaneously. Further, stereo images and red/blue three-dimensional images may be generated using the accompanying DESIPLOT program and an HP-7475A plotter. A version of the program is also currently available for the Apple Macintosh. Full implementation on the Macintosh requires 512 K and at least one disk drive. Otherwise this version is essentially identical to the IBM-PC version described herein.

The nucleocapsid protein from the Rous sarcoma virus has two regions of sequence with the motif Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Cys. All retrovirus nucleocapsid proteins contain one or two of these motifs, and they represent the only conserved sequences among these proteins. Sequence analysis of nucleocapsid from avian myeloblastosis virus shows that it also contains two Cys-His sequences and, in fact, differs from the Rous sarcoma nucleocapsid protein only in three residues near the carboxyl terminus. The hypothesized role of the conserved cysteines and histidines as zinc ligands was tested experimentally. No tightly bound metal ions were detected for avian myeloblastosis nucleocapsid protein, and the molar amount of zinc in virions was less by a factor of 50 than that of the nucleocapsid protein. Added Zn2+ did not significantly affect nucleocapsid binding to poly(ethenoadenylic acid) or its secondary structure, as determined from circular dichroism. Nevertheless, the conserved cysteine and histidine residues of the Rous sarcoma (Prague-C strain) nucleocapsid protein are essential for fully functional virus, as shown by the fact that single-site substitutions of five of the six conserved cysteines and either of the two histidine residues blocked viral replication.

The Fc fragment of a human monoclonal IgG1 was compared with subfragments containing (a) the intact CH2 domain (CH2 fragment) or (b) the intact CH3 domain (pFc' and tFc' fragments). All fragments were reductively 13C-methylated and their resulting dimethyllysyl resonances characterized in 0.1 M KC1 as a function of pH by 13C NMR spectroscopy. Seven resonances were characterized for the 18 lysine residues of the Fc fragment, eight for the 12 lysines of the CH2 fragment, and five each for the 18 lysine residues of the Fc fragment, eight for the 12 lysines of the CH2 fragment, and five each for the 9 lysines of the pFc' and the 6 lysines of the tFc' fragments, respectively. The multiplicity of resonances indicates that the lysine residues in each fragment exist in a variety of microenvironments and that the fragments are all highly structured. The correspondence between 6 of the 12 or 13 perturbed lysine residues in the Fc fragment and the smaller subfragments indicates that the conformation of the CH2 and CH3 domains is largely unchanged in the smaller fragments. However, in addition to three lysines at the CH2-CH3 domain interface, whose environments were known to be disrupted in the smaller fragments, three or four lysine residues have somewhat different properties in the Fc fragment and in the subfragments, indicating that some local perturbations are induced in the domain structure in the subfragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Reductive methylation of hen egg-white (HEW) lysozyme with [13C]formaldehyde and NaCNBH3 and subsequent 13C NMR spectroscopy reveal resonances for each of the mono- and dimethyl derivatives of the six lysyl epsilon-amino groups and the NH2 terminus. Each resonance has a unique chemical shift, pKa, and chemical shift change upon deprotonation. The assignment of the resonances arising from the N alpha,N-dimethyl and N alpha-monomethyl NH2 terminus has been made as have resonance assignments for the two lysyl residues which crystallographic studies indicate are involved in ion pair interactions (Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C., and Rupley, J. A. (1972) in The Enzymes (Boyer, P. D., ed) 3rd Ed, Vol. 7, pp. 665-868, Academic Press, New York). One resonance, tentatively assigned to the lysine 1 (epsilon NH3+) which forms an ion pair with glutamic acid 7 (gamma COO-), has a highly perturbed chemical shift which shows a biphasic titration curve (N epsilon, N-dimethyl pKa values 10.0 and 2.6). A similar titration curve is observed for a N epsilon-monomethyl lysyl residue. The resonance for lysine 13 (epsilon NH3+), which forms an ion pair with the carboxyl terminus, leucine 129 (alpha COO-), has been assigned by removal of leucine 129 with carboxypeptidase whereupon only one pair of mono- and dimethyl lysyl resonances is greatly perturbed. The pKa of N epsilon,N-dimethyl lysine 13 is 9.3 in des-Arg-Leu-lysozyme in contrast to 9.8 for the intact enzyme. Thus, it appears that both of the intramolecular ion pairs predicted by x-ray crystallography exist in the solution structure of HEW lysozyme.

We have used phosphorus-31 nuclear magnetic resonance to determine intracellular pH in the cellular slime mold Dictyostelium discoideum. We devised an air-lift circulator to maintain the dense cell suspensions in a well-oxygenated and well-stirred state while causing minimal perturbation to the sample flowing through the detector coils. Cells continued to develop normally in this set-up. Spectra acquired under these conditions typically show two peaks in the inorganic phosphate region corresponding to pH values of 7.16 +/- 0.03 and 6.48 +/- 0.02. These peaks are believed to represent the mitochondrial and cytosolic compartments respectively, based on a comparison of these values with published data and the collapse of the two compartments upon addition of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone. Dictyostelium cells show a remarkable degree of intracellular pH homeostasis. Both mitochondrial and cytosolic pH remained unchanged as extracellular pH was varied from 4.3 to 8.1. There was also no apparent change in the pH of either compartment after up to 13.5 hours' development in suspension.

A variety of studies have shown that differentiation of Dictyostelium discoideum amoebae in the presence of cAMP is strongly influenced by extracellular pH and various other treatments thought to act by modifying intracellular pH. Thus conditions expected to lower intracellular pH markedly enhance stalk cell formation, while treatments with the opposite effect favor spores. To directly test the idea that intracellular pH is a cell-type-specific messenger in Dictyostelium, we have measured intracellular pH in cells exposed to either low extracellular pH plus weak acid or high extracellular pH plus weak base using 31P nuclear magnetic resonance (NMR). Our results show that there is no significant difference in intracellular pH (cytosolic or mitochondrial) between pH conditions which strongly promote either stalk cell or spore formation, respectively. We have also examined the effects of external pH on the expression of various cell-type-specific markers, particularly mRNAs. Some mRNAs, such as those of the prestalk II (PL1 and 2H6) and prespore II (D19, 2H3) categories, are strongly regulated by external pH in a manner consistent with their cell-type specificity during normal development. Other markers such as mRNAs D14 (prestalk I), D18 (prespore I), 10C3 (common), or the enzyme UDP-galactose polysaccharide transferase are regulated only weakly or not at all by external pH. In sum, our results show that modulation of phenotype by extracellular pH in cell monolayers incubated with cAMP does not precisely mimic the regulation of stalk and spore pathways during normal development and that this phenotypic regulation by extracellular pH does not involve changes in intracellular pH.

Conventional biochemical analyses have demonstrated significant alterations in high-energy phosphate metabolism during shock, but the time course of these changes cannot be followed in individual animals because these analyses are invasive and destructive. This study sought to evaluate the utility of 31P NMR as a means of following phosphorus metabolites under various conditions, including those designed to model the shocked state. Twenty adult albino rats were subject to a modified Wiggers' model of hemorrhagic shock lasting from 5 to 140 min. ATP was determined on extracts of the kidneys of each animal both by a biochemical assay and by integration of 31P NMR resonance signals. The equation for renal ATP content plotted versus time for enzymatically determined ATP was 1.79 - 0.0097x (r = 0.83, P less than 0.01) as compared to 1.76 - 0.0093x (r = 0.69, P less than 0.01) for NMR-determined ATP. Isolated, normal rat kidneys perfused with oxygenated, modified Krebs' solution while in the NMR spectrometer maintained normal ATP levels for several hours. ATP/ADP ratios were greater than those observed by conventional enzymatic analysis. Temporary anoxia, induced by substituting 100% N2 for 95% O2:5% CO2, resulted in decreases in ATP content, which reverted to normal with reinstitution of oxygenation. Intracellular pH changed in accordance with perfusate pH during anoxia. It is concluded that 31P NMR studies of the perfused rat kidney have immediate application for the nondestructive study of energy metabolism in shock and ischemia.

Retroviruses encode a small, basic nucleocapsid (NC) protein that is found complexed to genomic RNA within the viral particle. The NC protein appears to function not only in a histone-like manner in packaging the RNA into the particle but also in specifically selecting the viral genomic RNA for packaging. A cysteine-histidine (cys-his) region, usually composed of 14 amino acids and reminiscent of the 'zinc fingers' of transcription factors, is the only highly conserved sequence element among the retroviral NC proteins. This review discusses the biochemical properties of NC, and its possible role(s) in retroviral replication. We also speculate on how the biochemical properties may relate to its function in RNA recognition and packaging.