The isozyme-specific effects of cyclooxygenase-deficiency on bone in mice.

Prostaglandin E(2) (PGE(2)) plays a critical role in skeletal physiology and bone loss. PGE(2) production is regulated in vivo by at least two cyclooxygenase (COX) isozymes, COX-1 and COX-2. The purpose of this study was to investigate the in vivo effects of the selective deletion of COX-1 or COX-2 on bone mineral density (BMD), bone microarchitecture and bone strength in wild type (WT), COX-1(-/-) and COX-2(-/-) mice. Using a LUNAR PIXImus, BMD was measured in 18 (WT), 18 COX-1(-/-) and 16 COX-2(-/-) mice. COX-1(-/-) mice exhibited significantly higher BMD (0.0506 g/cm(2) +/- 0.0014 g/cm(2)) than either WT (0.0493 g/cm(2) +/- 0.0019, P < or = 0.05) or COX-2(-/-) (0.0473 g/cm(2) +/- 0.0034, P < or = 0.01) mice. COX-2(-/-) mice had significantly lower BMD than WT (P < or = 0.01) or COX-1(-/-) (P < or = 0.01). Flexure stress of the femurs, determined by breaking the bones with three-point bending, correlated with bone density. Although plasma levels of both Ca(2+) and PTH were comparable in wild type and COX-1(-/-) mice, both were elevated in COX-2(-/-) mice consistent with primary hyperparathyroidism. These studies suggest that COX enzymes are important regulators of BMD and bone strength in mice. The beneficial effect of absence of the COX-1 enzyme on skeletal parameters may be secondary to decreases in PGE(2). On the other hand, primary hyperparathyroidism and lower bone magnesium content may account for the lower BMD and impairments in bone strength of COX-2(-/-) mice. Further elucidation of the effects of the COX pathway on bone remodeling may provide important information on potential therapeutic targets for preventing and/or treating osteoporosis.

Theoretical indicators of enzyme reaction specificity from conserved information in amino acid sidechains.

Amino acid sequences for 11 acetohydroxy acid synthase (EC 4.1.3.18; AHS) polypeptides with experimentally established activity were chosen for computational comparisons to detect conserved local information associated with reaction specificity for each sequence. Windowed analysis by Pearson product moment cross-correlation of six amino acid sidechain properties revealed locally conserved segments common to all proteins with AHS activity. Seven information segments were detected in the same arrangement in sequences for the large subunit polypeptides of prokaryotes, and in the sequences for single polypeptides of eukaryotic AHS. The information segments were numbered 1-7 according to sequential position, and sequence features such as cofactor binding sites were defined for specific segments. Extension of the information segment analysis to seven other proteins of the pyruvate decarboxylase superfamily permitted use of the content and organization of information segments to recognize four classes of enzyme reaction specificity. Estimates of information entropy, based upon a state space defined by reaction specificity, directly reflected the known reaction complexity for all but one enzyme examined. Our data suggest that development of information-segment models for enzyme superfamilies may improve the accuracy of inferring protein activity from sequence.

Vectors of shannon information from fourier signals characterizing base periodicity in genes and genomes.

Equal Symbol Fourier Transforms (FTES), characterizing nucleotide periodicity, comprise components of 5-D vectors that define base-repeat properties of a genomic sequence. This report describes a conversion of the FTES signals to a common platform of Shannon information content to facilitate comparisons of periodic data with other measures of information for genes and genomes. The autocorrelation used to compute the discrete FTES formed the basis to define repeating bases in terms of conditional probabilities. We derived a vector equation to express the Shannon information content of a sequence in a way that preserves the distinct specificity of base repeat patterns characterized by FTES vectors. We suggest application of such information vectors to study the structure of information in genes, chromosomes, and genomes by chi(2) comparisons.

Channeling behavior and activity models for Escherichia coli K-12 acetohydroxy acid synthases at physiological substrate levels.

The channeling behavior of acetohydroxy acid synthases I and III (EC 4.1.3.18; AHAS) was studied by computer simulation of activities over a wide range of concentrations for the substrates pyruvate and 2-ketobutyrate. The ratios of reaction rates for both channels and three-dimensional plots of single-channel reaction rates versus substrate concentrations were introduced to compare the substrate channeling properties of the isozymes. Substrate ranges were identified in which AHAS I and III operated both channels, and in which they used only one. Kinetic constants were varied to simulate whether and how AHAS might be made channel-specific. Our study suggests that AHAS might be made channel-specific for acetolactate but not for acetohydroxybutyrate. We postulate specific physiological roles for AHAS I and III to support cell growth under conditions that vary the levels and balance of substrates.

A mechanism for valine-resistant growth of Escherichia coli K-12 supported by the valine-sensitive acetohydroxy acid synthase IV activity from ilvJ662.

Acetohydroxy acid synthase (EC 4.1.3.18; AHAS) isozymes I and III are expressed in Escherichia coli strain K-12 and, when inhibited by L-valine, cannot support cell growth. AHAS IV, expressed from mutation at ilvJ662, exhibits valine-sensitivity similar to that of AHAS III, yet AHAS IV does support cell growth in valine minimal medium. Rate equations were derived for AHAS III and AHAS IV reaction in crude extracts and for partially purified AHAS IV. Values of kinetic constants in these equations were determined in order to model a probable reaction mechanism. Computer modeling of initial velocity reactions at physiological substrate concentrations simulated consequences of valine-inhibition and revealed that AHAS IV synthesized AHB at a maximal rate over four times faster than AHAS III under these conditions. The simulations predicted that cells depending upon AHAS III for growth in valine minimal medium would accumulate higher levels of 2-ketobutyrate than cells using AHAS IV. Experiments on growth inhibition by valine revealed more than a five-fold difference in 2-ketobutyrate accumulation, thus confirming these predictions. These data support the hypothesis that valine inhibition of growth is a consequence of 2-ketobutyrate accumulation to toxic levels. We propose that the valine-inhibited AHAS IV activity prevents growth inhibition by keeping 2-ketobutyrate accumulation to a lower level than resulting from AHAS III activity.

Age-associated alterations in hepatic beta-adrenergic receptor/adenylate cyclase complex.

The effect of age on catecholamine regulation of hepatic glycogenolysis and on hepatic adenylate cyclase was studied in male rats up to 24 mo of age. Epinephrine and norepinephrine stimulated glycogenolysis in isolated hepatocytes at all age groups studied. Isoproterenol, however, stimulated glycogenolysis only at 24 mo. In isolated liver membranes, usual activators of adenylate cyclase increased the activity of the enzyme considerably more in membranes from 24-mo-old rats than in membranes from either 3- or 21-mo-old rats. The Mn2+-dependent activity of the cyclase was increased by 2.9-fold in 3-mo-old animals and approximately 5.7-fold in 24-mo-old rats, indicating a substantial age-dependent increase in the intrinsic activity of the catalytic unit. The density of the beta-adrenergic receptor, as measured by the binding of [125I]-iodocyanopindolol to plasma membranes, was 5-8 fmol/mg protein in rats aged 3-12 mo but increased to 19 fmol/mg protein in 24-mo-old rats. Computer-aided analysis of isoproterenol competition of the binding indicated a small age-dependent increase (from 30% at 3 mo to 43% at 24 mo) in the proportion of beta-receptors in the high-affinity state. These observations suggest that beta-receptor-mediated hepatic glycogenolysis in the aged rat is predicated upon increases in the density of beta-receptors as well as increased intrinsic activity of the catalytic unit of adenylate cyclase.