Fatty Acid-Elongating Activity in Rapidly Expanding Leek Epidermis.

A microsomal fatty acid elongase activity measured in epidermis of rapidly expanding leek (Allium porrum L.) was 10-fold higher in specific activity than preparations from store-bought leek. These preparations elongated acyl chains effectively using endogenous or supplied primers. Elongation of C20:0 was specifically inhibited by 2 [mu]M cerulenin, and labeling experiments with [3H]cerulenin labeled two polypeptides (65 and 88 kD). ATP was required for maximal elongase activity in expanding leaves but was lost in nonexpanding tissues. Both [14C]stearoyl-coenzyme A (CoA) and [14C]stearate were maximally elongated in the presence of ATP. Addition of fully reduced CoA, however, inhibited [14C]stearate elongation, suggesting that stearoyl-CoA synthesis was not a prerequisite for elongation. Furthermore, microsomes preincubated with [14C]stearoyl-CoA plus ATP resulted in loss of radiolabel from the acyl-CoA pool without a corresponding loss in elongating activity. The lack of correlation between elongating activity and the label retained in the putative acyl-CoA substrate pool suggests that acyl-CoAs may not be the immediate precursors for elongation and that ATP plays a critical, yet undefined, role in the elongation process. We propose that an ATP-dependent elongating activity may generate the long-chain fatty acids required for wax biosynthesis.

Purification and Characterization of Acetyl-Coenzyme A Carboxylase from Diclofop-Resistant and -Susceptible Lolium multiflorum.

Acetyl-coenzyme A carboxylase (ACCase) was purified >100-fold (specific activity 3.5 units mg-1) from leaf tissue of diclofopresistant and -susceptible biotypes of Lolium multiflorum. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified fractions from both biotypes contained a single 206-kD biotinylated polypeptide. The molecular mass of the native enzyme from both biotypes was approximately 520 kD. In some cases the native dimer from both biotypes dissociated during gel filtration to form a subunit of approximately 224 kD. The inclusion of 5% (w/v) polyethylene glycol 3350 (PEG) in the elution buffer prevented this dissociation. Steady-state substrate kinetics were analyzed in both the presence and absence of 5% PEG. For ACCase from both biotypes, addition of PEG increased the velocity 22% and decreased the apparent Km values for acetyl-coenzyme A (acetyl-CoA), but increased the Km values for bicarbonate and ATP. In the presence of PEG, the Km values for bicarbonate and ATP were approximately 35% higher for the enzyme from the susceptible biotype compared with the resistant enzyme. In the absence of PEG, no differences in apparent Km values were observed for the enzymes from the two biotypes. Inhibition constants (Ki app) were determined for CoA, malonyl-CoA, and diclofop. CoA was an S-hyperbolic (slope replots)-I-hyperbolic (intercept replots) noncompetitive inhibitor with respect to acetyl-CoA, with Ki app values of 711 and 795 [mu]M for enzymes from the resistant and susceptible biotypes, respectively. Malonyl-CoA competitively inhibited both enzymes (versus acetyl-CoA) with Ki app values of 140 and 104 [mu]M for ACCase from resistant and susceptible biotypes, respectively. Diclofop was a linear noncompetitive inhibitor of ACCase from the susceptible biotype and a nonlinear, or S-hyperbolic-I-hyperbolic, noncompetitive inhibitor of ACCase from the resistant biotype. For ACCase from the susceptible biotype the slope (Kis) and intercept (Kii) inhibition constants for diclofop versus acetyl-CoA were 0.08 and 0.44 [mu]M, respectively. ACCase from the resistant biotype had a Ki app value of 6.5 [mu]M. At a subsaturating acetyl-CoA concentration of 50 [mu]M, the Hill coefficients for diclofop binding were 0.61 and 1.2 for ACCase from the resistant and susceptible biotypes, respectively. The Hill coefficients for diclofop binding and the inhibitor replots suggest that the resistant form of ACCase exhibits negative cooperativity in binding diclofop. However, the possibility that the nonlinear inhibition of ACCase activity by diclofop in the enzyme fraction isolated from the resistant biotype is due to the presence of both resistant and susceptible forms of ACCase cannot be excluded.

The relationship of nitrogen source and in vivo nitrate reductase actiity to root formation in Euphorbia esula cell suspension cultures.

Root formation and in vivo nitrate reductase (NR) activity were determined in leafy spurge cell suspensions. Cells grown in B5 media with 1 mg L(-1) 2,4-D were transferred to B5 media without 2,4-D, but containing either high (92:8) or low (15:85) ratios of nitrogen as NO 3 (-) -N:NH 4 (+) -N. In older cell lines root formation occurred only in the low NO 3 (-) medium with =<30 roots per flask. In younger cell lines root numbers were greatest in the high NO 3 (-) medium (1000 to 3000 per flask). Cells grown in low NO 3 (-) medium were about one-third the final dry weight as those in high NO 3 (-) medium. Root length was consistently greater for cell lines of all ages in the low NO 3 (-) medium. Developmental profiles of NR activity were similar in cell lines of all ages, whether or not roots were formed. NR activity was lower, however, in cultures grown in low NO 3 (-) medium compared to high NO 3 (-) medium. There was no consistent relationship between NR activity and root initiation. Therefore, nitrate reductase does not appear to be a primary target for regulation of leafy spurge growth by chemical application.

Short-term regulation of hydroxymethylglutaryl coenzyme A reductase by reversible phosphorylation: modulation of reductase phosphatase in rat hepatocytes.

Hydroxymethylglutaryl CoA reductase catalyzes the limiting step in cholesterol synthesis in liver and other tissues. Beginning in 1973 studies with subcellular systems established that microsomal reductase is inactivated with ATP(Mg) and reductase kinase, and restored to full activity with phospho-protein phosphatase. By contrast reductase kinase is inactivated with phosphatase and reactivated with a second protein kinase (reductase kinase kinase). This bicyclic system has now been confirmed in terms of homogeneous enzyme components and by direct reversible phosphorylation with [gamma 32P]ATP in several laboratories. Short-term endocrine control of reductase and reductase kinase has been demonstrated in intact rat hepatocytes. Preincubation of cells with glucagon brought about a fall in the expressed activity of reductase and a rise in reductase kinase consistent with net phosphorylation of both enzymes. Total reductase levels were also severely depressed after glucagon. Addition of insulin to suspensions of hepatocytes had the reverse effect on expressed activity of reductase (elevated) and reductase kinase (depressed). Insulin also prevented the decay in total reductase activity. Since both protein kinases identified in this system are cAMP-insensitive, it was possible that hormonal signaling is mediated through the protein phosphatase that acts on both reductase kinase and reductase. In recent studies we have shown that the rate of activation of endogenous reductase in hepatocyte extracts (microsomes plus cytosol) is responsive to hormonal modulation. Pretreatment of hepatocytes with insulin increases apparent reductase phosphatase activity in extracts while glucagon diminishes the rate of reductase activation. HMG CoA is converted to mevalonate by the reductase enzyme. In hepatocytes mevalonate is rapidly converted to cholesterol and to a variety of isoprene derivatives. Expressed reductase activity falls precipitously when hepatocytes are incubated with mevalonate (added in the form of mevalono-lactone). As in the case with glucagon pretreatment reductase phosphatase is rapidly diminished. (Mevalonate itself is not inhibitory to reductase or reductase phosphatase activity in subcellular systems.) It is probable that a product of mevalonate metabolism generated in intact cells may act as a reductase phosphatase inhibitor. Among these added inorganic pyrophosphate inhibited reductase phosphatase at low concentrations.