Knowledge-based simulation of DNA metabolism: prediction of enzyme action

We have developed a knowledge-based simulation of DNA metabolismthat accurately predicts the actions of enzymes on DNA undera large number of environmental conditions. Previous simulationsof enzyme systems rely predominantly on mathematical models.We use a frame-based representation to model enzymes, substratesand conditions. Interactions between these objects are expressedusing production rules and an underlying truth maintenance system.The system performs rapid inference and can explain its reasoning.A graphical interface provides access to all elements of thesimulation, including object representations and explanationgraphs. Predicting enzyme action is the first step in the developmentof a large knowledge base to envision the metabolic pathwaysof DNA replication and repair. Received on February 1, 1990; accepted on October 2, 1990     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.     

The role of the epidermal growth factor-1 and hydrophobic stack domains of human factor IX in binding to endothelial cells.

To determine the function and specificity in factor IX of the first epidermal growth factor (EGF)-like domain and the eight-amino acid hydrophobic stack encoded by exon C (residues 39-46), these domains were replaced by the corresponding polypeptide regions of factor X and chimeric proteins were produced in human embryo kidney cells. Both chimeras were activated by factor XIa at a rate similar to plasma factor IX and exhibited calcium-dependent fluorescence quenching similar to plasma factor IX. Both chimeras competed equally for binding to the endothelial cell receptor. Our findings make it unlikely that the first EGF-like domain or the hydrophobic stack of factor IX are responsible for the specific binding of factor IX to its endothelial cell receptor.     

Mu-opioid-receptor-mediated inhibition of the N-type calcium-channel current.

The predominant consequences of mu-opioid-receptor activation are depression of both neuronal activity and transmitter release. Mu-Opioid agonists have previously been observed to increase a potassium conductance and to inhibit adenylate cyclase. We now report that activation of mu-opioid receptors directly decreases the N-type calcium-channel current in a differentiated, human neuroblastoma cell line (SH-SY5Y). The coupling between the mu-opioid receptor and the calcium channel involves a pertussis toxin-sensitive G protein and is independent of changes in adenylate cyclase activity. The inhibition of the calcium-channel current is voltage dependent because it is largely overcome by strong membrane depolarization. It is not associated with changes in the kinetics of current inactivation. Therefore, the mu-receptor belongs to the superfamily of G-protein-coupled, inhibitory neurotransmitter receptors which modulate the activity of calcium and potassium channels and adenylate cyclase.     

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).     

Displacement of hepatic ornithine carbamoyltransferase from mitochondria to cytosol in Reye's syndrome.

In two patients with fatal Reye's Syndrome, total ornithine carbamoyltransferase (OCTase) activity in the liver was 50 and 75% of that found in three control livers. The levels of enzymatic activity would not be expected to have resulted in the 7- and 17-fold elevations in plasma ammonia levels found in the patients. Levels of 47 and 60% of the OCTase activity, however, were found in the cytosolic fraction compared to an average of 7% for control livers. Thus, the amount of enzymatic activity in the mitochondrial fractions was only 20 and 30% of that found in control mitochondrial fractions. This study suggests that, if only mitochondrial OCTase is active in the urea cycle, the decreases in functional enzyme found in Reye's Syndrome may be considerably greater than that reflected in total enzyme assays.