Zinc requirements of broiler breeder hens.

One hundred and twenty Cobb 500 hens, 20 wk of age, were randomly allocated into individual cages with the objective of estimating their Zn requirements. The study was composed of 3 phases: adaptation to cages (basal diet), depletion (deficient diet containing 18.7 ± 0.47 ppm Zn) for 7 wk, and experimental phases. Hens were fed diets with graded increments of Zn sulfate heptahydrate (ZnSO4·7H2O), totaling 18.7 ± 0.47, 50.3 ± 10.6, 77.3.0 ± 11.0, 110.2 ± 12.8, 140 ± 12.2, and 170.6 ± 13.2 ppm analyzed Zn in feeds for 12 wk (experimental phase). Requirements of Zn were done using quadratic polynomial (QP), broken line quadratic (BLQ), and exponential asymptotic (EA) models. In general, the non-linear statistical models were the ones that best fit the results in this study. Requirements obtained for hen day egg production and settable egg production were 83.3, 78.6 ppm and 61.4, 65.4 ppm for period of 33 to 36 wk, and 63.3, 53.1 and 60.4, 46.1 ppm for period of 37 to 40 wk, and 62.8, 52.8, and 67.7, 62.1 ppm for period of 41 to 44 wk, respectively, using BLQ and EA models. Total eggs and total settable eggs produced per hen had Zn requirements estimated as 75.7, 64.7 ppm, and 56.5, 41.5 ppm, respectively, for BLQ and EA models, whereas for alkaline phosphatase and eggshell percentage were 161.8, 124.9 ppm and 126.1, 122.4 ppm, using QP and BLQ models. Maximum responses for Zn in yolk for periods of 37 to 40 and 41 to 44 wk were 71.0, 78.1 and 64.5, 59.6 ppm, respectively, using BLQ and EA models. Breaking strength had Zn requirements estimated at 68.0 and 96.7 ppm, whereas eggshell palisade layer and eggshell thickness were maximized with 67.9, 67.9 ppm, and 67.7, 64.4 ppm, respectively, for BLQ and EA models. The average of all Zn requirement estimates obtained by EA and BLQ models in the present study was 72.28 ppm or 11.1 mg/hen/d.

Copper requirements of broiler breeder hens.

One-hundred-twenty Cobb 500 hens, 20 wk of age, were randomly allocated into individual cages with the objective of estimating Cu requirements. After being fed a Cu deficient diet for 4 wk, hens were fed diets with graded increments of supplemental Cu (0.0; 3.5; 7.0; 10.5; 14; and 17.5 ppm) from Cu sulfate (CuSO4 5H2O), totaling 2.67; 5.82; 9.38; 12.92; 16.83; and 20.19 ppm analyzed Cu in feeds for 20 weeks. Estimations of Cu requirements were done using exponential asymptotic (EA), broken line quadratic (BLQ), and quadratic polynomial (QP) models. Obtained Cu requirements for hen d egg production and total settable eggs per hen were 6.2, 7.3, and 12.9 ppm and 8.1, 9.0, and 13.4 ppm, respectively, using EA, BLQ, and QP models. The QP model was the only one having a fit for total eggs per hen with 13.1 ppm Cu as a requirement. Hemoglobin, hematocrit, and serum Cu from hens had requirements estimated as 13.9, 11.3, and 18.5, ppm; 14.6, 13.0, and 19.0 ppm; and 16.2, 14.6, and 14.2 ppm, respectively, for EA, BLQ, and QP models. Hatching chick hemoglobin was not affected by dietary Cu, whereas requirements estimated for hatching chick hematocrit and body weight and length were 10.2, 12.3, and 13.3 ppm using EA, BLQ, and QP models; and 6.8 and 7.1 ppm, and 12.9 and 13.9 ppm Cu using EA and BLQ models, respectively. Maximum responses for egg weight, yolk Cu content, and eggshell membrane thickness were 14.9, 12.7, and 15.1 ppm; 15.0, 16.3, and 15.7 ppm; and 7.3, 7.8, and 14.0 ppm Cu, respectively, for EA, BLQ, and QP models. Yolk and albumen percentage were adjusted only with the QP model and had requirements estimated at 11.0 ppm and 11.3 ppm, respectively, whereas eggshell mammillary layer was maximized with 10.6, 10.1, and 14.4 ppm Cu using EA, BLQ, and QP models, respectively. The average of all Cu requirement estimates obtained in the present study was 12.5 ppm Cu.

Stringent and relaxed specificities of TaqI endonuclease: interactions with metal cofactors and DNA sequences.

We have studied the roles of metal cofactors Mg2+ and Mn2+ in modulating substrate specificities during the enzymatic cycle of TaqI endonuclease using steady state and single-turnover kinetics. In the presence of Mg2+, stringent discrimination of TaqI against single base-pair changes (star sites) is manifested by the loss of tight, specific binding in the early stage of the enzymatic cycle. In the presence of Mn2+, relaxed specificity for a star site sequence is attributed to formation of three distinct classes of the ternary complexes: the highly activated TaqI-cognate-Mn2+ complex; the partially activated TaqI-star-Mn2+ complex; and the ground state, inactive TaqI-nonspecific-Mn2+ complex. In addition to a high affinity for a TaqI-DNA complex, Mn2+ also binds to TaqI in a DNA-independent fashion. This may facilitate enzyme activation, which could account for the observed relaxation in substrate specificity. Thus, the TaqI-DNA-Mn2+ complex could be formed by either of two pathways: TaqI binding to DNA followed by the binding of Mn2+ or TaqI first binding to Mn2+ followed by the addition of DNA. The inactive, nonspecific TaqI-star-Mg2+ complex virtually prohibits transition state interactions, but a TaqI-star-Mn2+ complex attains a measurable single-turnover rate. In the late stages of the enzymatic cycle, high affinity of Mn2+ to a TaqI-DNA complex and to the TaqI enzyme may also account for a slower rate of product release.

Photoaffinity cross-linking of TaqI restriction endonuclease using an aryl azide linked to the phosphate backbone.

In an effort to identify amino acid (aa) residues near the active site of TaqI restriction endonuclease (ENase), a sequence-specific photoaffinity reagent was designed. This reagent exploits the finding that modification of the Rp oxygen of the scissile phosphate does not interfere with substrate binding. The TpCGA phosphate was substituted with an Rp phosphorothioate group to direct the placement of the heterobifunctional reagent p-azidophenacyl bromide. TaqI bound the photoaffinity reagent specifically and formed a covalent adduct with the ENase in the presence of UV light. The modified aa was identified as Tyr161. This aa was changed to Phe by site-directed mutagenesis, and the resulting Y161F mutant was characterized. Removal of the Tyr161 hydroxyl group lowered both the kcat and the Km fivefold, indicating that, while this aa may be near the scissile phosphate, it is not critically required for catalysis.

Interaction of TaqI endonuclease with the phosphate backbone. Effects of stereospecific phosphate modification.

The restriction endonuclease TaqI exhibits extreme specificity for its cognate sequence, TCGA, but a direct hydrogen bond readout model fails to account for this property. The present study examines the role of phosphate contacts in the enzyme-substrate and transition state complexes. An S-methyl group was introduced into each of the pTpCpGpApNpN internucleotide linkages using a hybrid chemical-enzymatic synthesis, in which sulfur substitutions of nonbridging phosphate oxygens directed the placement of methyl groups. The resulting 12 diastereomerically pure phosphate-modified substrates were tested for binding and cleavage by TaqI. The largest binding effects were induced by pro-Sp methylations at the pTpCpGA phosphates, which destabilized the enzyme-substrate complex by 1.0-1.6 kcal/mol. Cleavage of the modified strand was inhibited completely by modifications at the TpCpGpA phosphates and inhibited significantly at the TCGApNp phosphates. Cleavage of both strands was completely inhibited by modification of the TCGpA linkage. Effects on the cleavage of the unmodified strand were used to implicate phosphate modifications that caused global perturbations in the structure of the transition state complex. These results lend support for a model for the specificity of TaqI, in which sequence-specific phosphate contacts are formed in the transition state, thus amplifying the apparent contribution of base contacts to transition state stabilization.

Detection, resolution, and nomenclature of multiple ubiquitin carboxyl-terminal esterases from bovine calf thymus.

In vivo, ubiquitin exists both free and conjugated through its carboxyl terminus to the alpha- and epsilon-amino groups of a wide variety of cellular proteins. Ubiquitin carboxyl-terminal hydrolytic activity is likely a necessary step in the regeneration of the ubiquitin cofactor from ubiquitin-protein conjugates. In addition, this type of activity is required to generate the active, monomeric ubiquitin from the only known gene products: the polyprotein precursor and various ubiquitin fusion proteins. Thus, this activity is of vital importance to systems that utilize ubiquitin as a cofactor. A generic substrate, ubiquitin ethyl ester, was previously developed [Wilkinson, K. D., Cox, M. J., Mayer, A. N., & Frey, T. (1986) Biochemistry 25, 6644-6649] and utilized here to monitor the fractionation of these activities from calf thymus. By use of a rapid HPLC assay, four distinct, ubiquitin-specific esterases were identified and separated. A previously undescribed activity has been resolved and characterized, in addition to the bovine homologue of ubiquitin carboxyl-terminal hydrolase purified from rabbit reticulocytes. Two other activities resemble deconjugating activities previously detected in crude extracts but not previously purified. These activities appear to form a family of mechanistically related hydrolases. All four activities are inhibited by iodoacetamide, indicating the presence of an essential thiol group, and are inhibited to various extents by manganese. All have specific ubiquitin binding sites as judged by the low observed Km values (0.6-30 microM). The carboxyl-terminal aldehyde of ubiquitin is a potent inhibitor of these enzyme activities, with Ki values approximately 1000-fold lower than the respective Km values.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol-induced conformational changes of ubiquitin.

Ubiquitin has been found to be soluble in ethylene glycol and alcohols as the perchlorate or hydrochloride salt. When the effect of alcohol on the structure of ubiquitin is examined, two reversible conformational transitions are observed. Upon lowering the dielectric constant of aqueous alcohol solutions of ubiquitin from 80 to 45, the native structure of ubiquitin is converted to a form consistent with 50% helical structure. This conformational change results in a change in exposure to solvent of the single methionine and the single tyrosine residues of ubiquitin. In agreement with crystallographic results, these residues are buried in the native conformation but become fully exposed to solvent upon undergoing this transition. Further lowering of the dielectric constant to 20 results in the accumulation of a conformation with almost complete helical structure. Thus, hydrophobic interactions cause facile conformational changes in the ubiquitin structure. These results are discussed in terms of a preferential solvation model. It is shown that the results obtained with different alcohols can be normalized by the use of a dielectric constant scale. This normalization corrects for the different molar volumes of different alcohols, allows comparison of results obtained with different alcohols, and should be useful in studying this phenomenon with different proteins.

Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase.

A new substrate for ubiquitin carboxyl-terminal hydrolase, the carboxyl-terminal ethyl ester of ubiquitin, has been synthesized by a trypsin-catalyzed transpeptidation. In the presence of 1.6 M glycylglycine ethyl ester, trypsin removes the carboxyl-terminal glycylglycine of ubiquitin and replaces it with the dipeptide ester. The equilibrium mixture under these conditions contains 30% ubiquitin ethyl ester and 70% hydrolysis product, the 74-residue fragment of ubiquitin. Ubiquitin ethyl ester can be purified by gel filtration and ion-exchange chromatography. The structure of this product has been verified by identification of the products of base hydrolysis, tryptic cleavage in aqueous solution, and peptide mapping. When ubiquitin ethyl ester is incubated with purified ubiquitin carboxyl-terminal hydrolase, specific cleavage of the ester linkage is observed. A rapid, sensitive assay is described utilizing high-performance liquid chromatography. By use of this assay, it has been shown that ubiquitin carboxyl-terminal hydrolase is inactivated in the absence of thiols. Optimal protective effects are seen with 10 mM dithiothreitol. The rate of catalysis is maximal at pH 8.5, with evidence for catalytically important groups with pK values of 5.2, 7.6, and 9.5. These findings are consistent with the participation of a thiol group in the active site. Native ubiquitin is a competitive inhibitor of ubiquitin ethyl ester hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)