Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: a model for the analysis of multi-domain proteins.

We have designed and constructed a DNA sequence encoding human tissue plasminogen activator (tPA) with convenient restriction sites that flank each of the domains of the heavy chain. To accomplish this, the first 1095 bases of the gene coding for the mature protein were synthesized with unique restriction sites engineered into the interdomainal regions. This synthetic construction was then ligated to a cDNA fragment of the tPA gene that encoded the active site, thus generating a full-length tPA gene. The gene products produced by Chinese hamster ovary (CHO) cells transfected with either the tPA cassette gene or the tPA cDNA gene were then compared with the tPA produced by Bowes melanoma cells to determine whether or not synthetic interdomainal amino acid changes had an effect on the biochemical characteristics of the molecule. Specifically, molecular weight, specific activity, enhancement by fibrinogen fragments and kinetic constants were analysed. None of the properties examined were significantly different from those of the native melanoma tPA. Therefore, the cassette gene described herein should provide considerable versatility and precision in the construction of tPA mutants by facilitating the manipulation of the finger, growth factor and kringle domains, and likewise should be useful in assessing the function of these domains within the tPA molecule. We present this cassette gene system as a model for the analysis of protein domain function applicable to other multi-domain proteins.

Evidence that chlorpromazine inhibits sterologenesis at post-HMGCoA reductase sites in rat liver, in vitro.

The mechanism by which chlorpromazine inhibits cholesterogenesis in rat liver was investigated in vitro with the use of [14C] acetate and [14C] mevalonate as sterol precursors. Evidence was obtained that chlorpromazine blocks cholesterogenesis at multiple sites beyond HMGCoA reductase (beta-hydroxy-beta-methylglutarylCoA reductase, EC 1.1.1.34), the rate-limiting step. Squalene synthesis from both labeled acetate and mevalonate is reduced to a similar extent in the presence of chlorpromazine (29-36% at 0.5 mM). The data indicate that there is also an impairment of conversion of squalene to lanosterol, and of lanosterol to cholesterol. Overall inhibition of cholesterogenesis by chlorpromazine reached 65-75% at 0.5 mM and was concentration-dependent over the range 0.15-1.0 mM.

Stimulation of hepatic squalene and triglyceride synthesis by dimethylsulfoxide, in vitro.

The incorporation of [14C]mevalonate and [14C]acetate into squalene by rat liver slices was increased over 7-fold by the presence of 5% dimethylsulfoxide (DMSO) in the incubation medium. The stimulation of squalene synthesis was dose-related over the concentration range of 1-5% DMSO and did not affect the incorporation of [14C]mevalonate into the C27-sterol fraction (cholesterol) but did increase (about 50%) incorporation into C30-sterol (lanosterol) at a level of 5% DMSO. The stimulation of squalene synthesis was observed under both anaerobic (N2 atmosphere) and aerobic (ambient air or 95% O2/5% CO2) conditions and may represent a direct effect of DMSO on squalene synthetase. At a level of 5%, DMSO also stimulated 7-fold the incorporation of [14C]acetate into triglycerides by liver slices; this occurred without changes in incorporation into the phospholipid or free fatty acid fractions. The disproportionate increase in lipid labeling from [14C]acetate suggests that the effects of DMSO are not simply a matter of increasing [14C]acetate entry into the tissue.

Effect of chlorpromazine on rat arterial lipid synthesis, in vitro.

The effect of chlorpromazine, a major tranquilizer, on arterial lipid metabolism was studied in vitro in rat aortas incubated with [14C]acetate and [14C]mevalonate as lipid precursors. Chlorpromazine at a level of 0.25 mM in the incubation medium significantly reduced the incorporation of [14C]acetate into free fatty acids (p less than 0.01) and total phospholipids (p less than 0.001) but not triglycerides. Chlorpromazine also altered the pattern of arterial phospholipids synthesized from [14C]acetate by significantly increasing the relative proportion of phosphatidylinositol plus phosphatidylserine (p less than 0.02) and reducing the relative proportion of sphingomyelin (p less than 0.001). [14C] Acetate incorporation into the combined fractions of steryl esters plus hydrocarbons and sterols plus diglycerides was also significantly reduced (p less than 0.001) by 0.25 mM chlorpromazine. Studies with [14C]mevalonate showed that chlorpromazine is also an inhibitor of sterol biosynthesis in arterial tissues as evidenced by 35-40% reductions (p less than 0.05) in the formation of 14C-labeled squalene and C27 sterols.

Inhibition of LCAT in plasma from man and experimental animals by chlorpromazine.

Chlorpromazine (CPZ), a major tranquilizer, was found to be a potent inhibitor of lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) in the plasma of normal man, rat, rabbit and dog in vitro. The inhibitory effect of CPZ reached 35-50% at 0.5 mM depending on species; dog plasma LCAT appeared to be somewhat more sensitive than that of the other species. In rats fed CPZ or lidocaine for 14 days (0.05% in the diet), there was no statistically significant change in total plasma cholesterol levels or the size of the plasma-free (unesterified) cholesterol pool. However, 5 hr after an intracardial injection of [14C]cholesterol, the percentage of plasma [14C]cholesterol that was esterified was significantly lower (ca. 6%, p less than 0.05) in the CPZ-treated group, suggesting that CPZ may also inhibit LCAT To some extent in vivo. The percentage of plasma [14C]cholesterol esterified in the lidocaine-treated group was similar to control values and did not reflect its ability to inhibit LCAT in vitro.

Membrane-active agents. Effect of various anesthetics and chlorpromazine on arterial lipid metabolism.

The local anesthetic lidocaine was studied for its effects on lipid metabolism in aortas from normal rats, rabbits, and cholesterol-fed (atherosclerotic) rabbits in vitro. Incubation of aortas in the presence of 3--5 mM lidocaine resulted in a statistically significant reduction in the incorporation of [14C]oleate into cholesteryl esters and phosphatidylcholine. Additionally, significant increases in [14C]oleate incorporation into the diglyceride fraction of atheromatous rabbit aortas was observed with a trend to greater incorporation into the diglyceride fraction of normal rat and rabbit arteries as well. The most significant overall effect of lidocaine was its inhibition (50--90%) of the arterial sterol esterification. Assays of acylCoA : cholesterol acyltransferase (ACAT, EC 2.3.1.26) in isolated arterial microsomes revealed that, in addition to local anesthetics (e.g., lidocaine), other membrane-active agents such as chlorpromazine and methoxyflurane inhibit ACAT; this suggests ACAT may be regulated by alterations in the biophysical properties of its membrane milieu.

Lecithin-cholesterol acyltransferase: inhibition by local anesthetics in plasma from man and experimental animals in vitro.

Lecithin-cholesterol acyltransferase (LCAT, EC 2.3.1.43) was assayed in vitro in plasma from normal man, rat, rabbit and dog by following the formation of 14C-cholesteryl esters subsequent to labeling the plasma with 14C-cholesterol in vitro. In all species examined, various local anesthetics were found to inhibit LCAT when studied over the concentration range of 1 to 5 mM. The order of inhibition was dibucaine>benzocaine>tetracaine>lidocaine>procaine. Since LCAT activity represents the combined effect of a deacylation step and an esterification step, inhibition of LCAT by local anesthetics could theoretically involve either or both steps.

Effect of local anesthetics on sterol biosynthesis and sterol esterification in rat liver in vitro.

Various local anesthetics were found to inhibit sterol esterification by acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26) in the microsomal fraction from rat (and rabbit) liver and to inhibit sterologenesis in rat liver slices. The enzyme was assayed in isolated microsomes by following the incorporation of [1-14C]oleoyl-CoA into steryl esters; the order of inhibitory potency on the enzyme was dibucaine > benzocaine > tetracaine > lidocaine > procaine. In liver slices, the incorporation of labeled acetate and mevalonate into C27 sterols, digitonin-precipitable sterols, and squalene was inhibited in the order dibucaine > tetracaine > lidocaine. A comparison of incorportion patterns from labeled acetate and mevalonate suggest that inhibition of sterologenesis occurs at more than one post-mevalonate step in the pathway.