Can a signal sequence become too hydrophobic?

We have characterized several mutants that contain alterations in the hydrophilic domain (N region) of the pseudorabies virus glycoprotein gC signal sequence. In general, our results agree with previous findings and indicate that basic residues in the N region are not essential for efficient export of gC in infected cells. While reducing the N region to a net neutral charge led to a slight impairment of membrane translocation, a substantial gC export defect was not observed until a net negative charge was introduced. However, there was one exception to this pattern. The substitution of a leucine for an arginine at the carboxyl terminus of the N region led to a considerable export defect despite maintaining a net positive charge. As a consequence of the substitution, the mutant signal sequence was 1.5 times more hydrophobic than wild type, but we found that the defect could be largely corrected if an additional alteration that lessened the overall hydrophobicity of the gC signal sequence was incorporated. We suggest that an upper limit of hydrophobicity may exist for eukaryotic signal sequences; exceeding this value could lead to an export defect.

Immunomodulation and drug acetylation: influence of the immunomodulator tilorone on hepatic, renal and blood N-acetyltransferase activity and on hepatic cytosolic acetyl coenzyme A content.

The biochemical alteration responsible for immunomodulator enhancement of drug acetylation in vivo was probed ex vivo and in vitro in the rat. Rat liver or kidney cytosol, obtained by differential centrifugation, or whole blood served as the source of N-acetyltransferase (NAT). Addition of tilorone (0.5-8.0 mM) to incubation mixtures containing procainamide (PA, 0.6 mM) and acetyl coenzyme A (AcCoA, 0.42 mM) resulted in the inhibition of N-acetylprocainamide formation, while lower concentrations of tilorone had no effect. Pretreatment of rats with tilorone (50 mg/kg) administered orally 48 hr prior to sacrifice did not alter hepatic apparent Km and Vmax for NAT toward PA compared to control animals. Utilization of an AcCoA regenerating system in the incubation mixtures also resulted in no significant differences in the apparent Michaelis-Menten parameters obtained. Acetylation activity in kidney and whole blood also was not altered by immunomodulator pretreatment. Hepatic cytosolic AcCoA content was reduced significantly 48 hr after tilorone pretreatment (5.10 +/- 2.1 vs 11.97 +/- 2.2 nmol/mg protein) (P less than 0.05). These data indicate that an increase in NAT content or activity is not the biochemical alteration responsible for immunomodulator enhancement of drug acetylation, and that the required cofactor, cytosolic AcCoA, is decreased by immunomodulator pretreatment.

Effect of H2-receptor antagonists on rat liver cytosolic acetyl CoA:arylamine N-acetyltransferase activity.

This investigation examined the effect of cimetidine, famotidine, and ranitidine on rat liver acetyl CoA:arylamine N-acetyltransferase (NAT) activity. Studies were conducted using procainamide and p-aminobenzoic acid as substrate probes for NAT isozymes II and I, respectively. At an inhibitor:substrate ratio of 2:1, ranitidine, cimetidine, and famotidine reduced NAT II activity by 9, 48, and 75%, respectively. At this same ratio, none of the H2-receptor antagonists significantly reduced NAT I activity. The inhibition of NAT II activity by cimetidine and famotidine was mixed in nature, with characteristics consistent with predominantly competitive inhibitors. Preincubation of NAT with acetyl CoA did not attenuate the inhibitory effects of famotidine, suggesting this agent does not associate with the sulfhydryl of the critical cysteine residue on NAT. These results indicate the ability of H2-receptor antagonists to inhibit NAT activity with some degree of specificity for the two isozymes and significant differences in inhibitory potency between the antagonists.

Effects of chloroquine and primaquine on rat liver cytosolic N-acetyltransferase activity.

Chloroquine caused only slight reductions in NAT activity when added in vitro, and had no detectable influence when animals were pretreated with it for 4 days. This would suggest that the previously reported reduced excretion of acetylated metabolites of INH and SDD following chloroquine pretreatment is not the result of inhibition of NAT. In contrast, we found that primaquine significantly (P less than 0.05) reduced NAT activity when added in vitro, suggesting the need for further study with this agent.