Generation of an active monomer of rabbit muscle creatine kinase by site-directed mutagenesis: the effect of quaternary structure on catalysis and stability

Cytosolic creatine kinase exists in native form as a dimer; however, the reasons for this quaternary structure are unclear, given that there is no evidence of active site communication and more primitive guanidino kinases are monomers. Three fully conserved residues found in one-half of the dimer interface of the rabbit muscle creatine kinase (rmCK) were selectively changed to alanine by site-directed mutagenesis. Four mutants were prepared, overexpressed, and purified: R147A, R151A, D209A, and R147A/R151A. Both the R147A and R147A/R151A were confirmed by size-exclusion chromatography and analytical ultracentrifugation to be monomers, whereas R151A was dimeric and D209A appeared to be an equilibrium mixture of dimers and monomers. Kinetic analysis showed that the monomeric mutants, R147A and R147A/R151A, showed substantial enzymatic activity. Substrate binding affinity by R147A/R151A was reduced approximately 10-fold, although k(cat) was 60% of the wild-type enzyme. Unlike the R147A/R151A, the kinetic data for the R147A mutant could not be fit to a random-order rapid-equilibrium mechanism characteristic of the wild-type, but could only be fit to an ordered mechanism with creatine binding first. Substrate binding affinities were also significantly lower for the R147A mutant, but k(cat) was 11% that of the native enzyme. Fluorescence measurements using 1-anilinonaphthalene-8-sufonate showed that increased amounts of hydrophobic surface area are exposed in all of the mutants, with the monomeric mutants having the greatest amounts of unfolding. Thermal inactivation profiles demonstrated that protein stability is significantly decreased in the monomeric mutants compared to wild-type. Denaturation experiments measuring lambda(max) of the intrinsic fluorescence as a function of guanidine hydrochloride concentration helped confirm the quaternary structures and indicated that the general unfolding pathway of all the mutants are similar to that of the wild-type. Collectively, the data show that dimerization is not a prerequisite for activity, but there is loss of structure and stability upon formation of a CK monomer.     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

Hematology and blood biochemistry in infant baboons (Papio hamadryas)

Although published normative reference standards for hematologic and clinical chemistry measures are available for adult baboons, their applicability to infants has not been addressed. We analyzed these measures in 110 infant baboons (55 females and 55 males) from a large breeding colony at the Southwest Regional Primate Research Center in San Antonio, Texas. The sample consists of olive baboons and olive/yellow baboon hybrids, 1 week to 12 months of age. We produced cross-sectional reference values and examined the effects of age, sex, and subspecies on these variables. Hematology reference ranges for infant baboons are similar to, but wider than, those for adults. Reference ranges for blood biochemistry measures are generally more dissimilar to adults, indicating that for many variables, reference ranges for adult baboons are not adequate for infants. Although sex and subspecies differences are rare, age accounts for more than 10% of the variance in many of the variables.     

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.     

The correlation of protein structure and evolution of a protein-coding gene: phylogenetic inference using cytochrome oxidase III

Discriminating phylogenetic signal from noise in DNA sequence data is a difficult problem in phylogenetic inference at higher systematic levels. For protein-coding genes, noise at synonymous (silent) positions can be filtered by deleting entire codon positions or types of change at a codon position. This method is not appropriate for replacement sites, because changes at each site within a codon may not be independent. This research presents a method using information from protein structure to evaluate variation in replacement sites. Analysis of the correlation of amino acid variation with protein structure identified rapidly evolving codons in the COIII gene. In a series of phylogenetic analyses attempting to recover a known set of vertebrate relationships, downweighting these labile codons produced the most accurate results. Structural correlates of variable and invariant residues identified in this study can be used to increase the accuracy of models used for phylogenetic inference. Viewing amino acid variation within a phylogenetic framework provided insight into residue changes important in the secondary and tertiary structures of the molecule, changes that were correlated between pairs of neighboring residues or between residues in neighboring helices.      

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Genotypic differences in thermotolerance are dependent upon prestress capacity for antioxidant protection of the photosynthetic apparatus in Gossypium hirsutum

Numerous studies have illustrated the need for antioxidant enzymes in acquired photosynthetic thermotolerance, but information on their possible role in promoting innate thermotolerance is lacking. We investigated the hypothesis that genotypic differences in source leaf photosynthetic thermostability would be dependent upon prestress capacity for antioxidant protection of the photosynthetic apparatus in Gossypium hirsutum. To test this hypothesis, thermosensitive (cv. ST4554) and reportedly thermotolerant (cv. VH260) G. hirsutum plants were exposed to control (30/20°C) or high‐day temperature (38/20°C) conditions during flowering and source leaf gas exchange, chlorophyll content and maximum photochemical efficiency (F_v/F_m) were measured for each treatment. The relationship between source leaf thermostability and prestress antioxidant capacity was quantified by monitoring the actual quantum yield response of photosystem II (PSII) (Φ_PSII) to a range of temperatures for both cultivars grown under the control temperature regime and measuring antioxidant enzyme activity for those same leaves. VH260 was more thermotolerant than ST4554 as evidenced by photosynthesis and F_v/F_m being significantly lower under high temperature for ST4554 but not VH260. Under identical growth conditions, VH260 had significantly higher optimal and threshold temperatures for Φ_PSII and glutathione reductase (GR; EC 1.8.1.7) activity than ST4554, and innate threshold temperature was dependent upon endogenous GR and superoxide dismutase (SOD; EC 1.15.1.1) activity. We conclude that maintaining a sufficient antioxidant enzyme pool prior to heat stress is an innate mechanism for coping with rapid leaf temperature increases that commonly occur under field conditions.