Specific activity

Abstract: The activity of leukotriene A4 (LTA4) synthase in crude human leukocyte homogenates was found to have a similar requirement for Ca2+ and ATP as had been noted previously for 5-lipoxygenase activity. Purification of the 5-lipoxygenase using ammonium sulfate fractionation, AcA 44 gel-filtration chromatography, and HPLC on anion-exchange and hydroxyapatite columns demonstrated that LTA4 synthase activity copurified with the 5-lipoxygenase with similar recoveries and increases in specific activity. Furthermore, the two enzymatic activities coeluted exactly on three different HPLC systems. Maximal activity of purified LTA4 synthase required the addition of three nondialyzable stimulatory factors, two of which were cytosolic and one of which was membrane-bound. These findings were identical for 5-lipoxygenase activity. When incubated with arachidonic acid, the purified 5-lipoxygenase converted approximately equal to 15% of its endogenously generated 5-hydroperoxyicosatetraenoic acid (5-HPETE) to LTA4. LTA4 production was more efficient when the enzyme utilized 5-HPETE generated from arachidonic acid than when 5-HPETE was exogenously supplied as substrate. These findings suggest that a single protein from human leukocytes possesses 5-lipoxygenase and LTA4 synthase activities and that the synthesis of LTA4 from 5-HPETE is controlled by the same complex multicomponent system that regulates the 5-lipoxygenase reaction.

Abstract: Protein kinase activity results in the incorporation of radiolabeled phosphate from [γ-32P]ATP into a peptide or protein substrate. The measurement of the amount of radioactivity incorporated into a substrate as a function of time and enzyme concentration allows enzyme activity to be quantified. The activity is expressed as a 'unit', where 1 unit corresponds to the amount of protein kinase that catalyzes the incorporation of 1 nanomole of phosphate into the standard substrate in 1 minute. Specific activity is defined as units of activity per milligram protein. The assay format described here is quick, simple, inexpensive, sensitive and accurate, provides a direct measurement of activity and remains the 'gold standard' for the quantification of protein kinase activity. Up to 40 samples can be assayed manually at one time, and the assay takes one person less than 1 hour to complete.

Abstract: We previously reported that cytosolic fractions of bovine brain contain two immunologically distinct forms of phospholipase C (PLC), PLC-I and PLC-II. We now report the purification of another form of inositolphospholipid-specific phospholipase C from bovine brain cytosol, designated PLC-III, and the comparison of the catalytic properties of the three isozymes. Approximately 450 micrograms of pure PLC-III was obtained from 36 bovine brains, and it had a final specific activity of 30-40 mumol of phosphatidylinositol hydrolyzed per min per mg of enzyme in the presence of 0.1% deoxycholate. PLC-III exhibited an apparent Mr of 85,000 in NaDodSO4/PAGE, which is considerably smaller than the Mr of 150,000 for PLC-I and 145,000 for PLC-II. Neither of the two mixtures of monoclonal antibodies nor the rabbit polyclonal antibodies directed against either PLC-I or PLC-II cross-reacted with PLC-III. The catalytic properties of the three isozymes were studied by using small unilamellar vesicles prepared from either phosphatidylinositol (PtdIns) or phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates. Hydrolysis of both PtdIns and PtdInsP2 by the three enzymes was dependent on Ca2+. However, at low Ca2+ concentration, PtdInsP2 was the preferred substrate for all three enzymes. When PtdIns was the substrate, the three enzymes exhibited similar specific activities at their optimum pH, which was 4.8 for PLC-I, 5.0 for PLC-II, and 5.5 for PLC-III. But at neutral pH, the order of specific activity was PLC-III greater than PLC-II greater than PLC-I. In contrast, the order of specific activity was PLC-I greater than PLC-III greater than PLC-II for PtdInsP2 hydrolysis, which means that PLC-I is the most specific for PtdInsP2. The three enzymes were affected differently by bovine serum albumin: inhibition of PLC-I and activation of PLC-III were observed, whereas PLC-II was unaffected. This observation suggests that any putative protein effectors for PLC should be critically scrutinized.