Phospholipase B Research Articles

Two degradation pathways of endoplasmic reticulum in fish hepatocytes

Zebrafish hepatocytes respond to stimulation with estradiol 17β (E2) with an extreme enlargement of the endomembrane system, especially the endoplasmic reticulum (ER), and when stimulation is stopped with a rapid degradation of enlarged endomembranes. Two pathways for degradation of ER were studied: a) the autophagy which was evaluated by stereological measurement; and b) the activity of cytosolic phospholipase B (PL-B) measured by a titration method. After a 30-day treatment with E2 a six-fold increase of the surface density of ER was accompanied by an increase of both autophagy and PL-B activity. 2 days after stopping the stimulation with E2 the ER vesiculated and its surface density decreased to the half value. Interestingly, at the same time autophagic vacuoles (AV) almost disappeared from hepatocytes, while the activity of PL-B reached its maximum at which it persisted for a further 4 days. After 4–6 days without E2 the cistern of ER became flattened again and new AVs reappeared. The data suggest that the regulation mechanisms of endomembrane degradation by PL-B and autophagy do not depend on each other and also that the appearance of AV is strongly related to the shape of ER.

Serine 228 is essential for catalytic activities of 85-kDa cytosolic phospholipase A2.

The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).

A modified method for experimental candidosis in mice avoiding lethality.

A model is presented which selected one out of 150 Candida albicans strains for the evaluation of antifungal agents. The mice were inoculated with 6 x 10(5) CFU of strain 352 into the tail vein. The strain has a moderate phospholipase B (PLB) activity in vitro and was originally isolated from a stool sample from a patient in an intensive care unit. This infection leads to very little suffering in the infected animals during the 6-day observation period. Kidney counts at day 5 after infection can give a first indication for a possible fungistatic mechanism. Possible interesting drugs can then be evaluated by a second set of experiments using a longer observation time to investigate the compounds for fungicidal properties. The model suggests that screening for systemic antifungals by avoiding lethality of mice in the first place can be done.

Phospholipase A and B activities of reptile and Hymenoptera venoms

Phospholipase B (PLB) activity was found in a variety of snake, Gila monster and Hymenoptera venoms. Hymenoptera venoms, except bee, are an especially rich source of PLB. This was shown by incubation of crude venoms with lysophosphatidylcholine and subsequent titration of liberated fatty acids. PLB activity was not found in pure α- or β-bungarotoxin, α-cobrotoxin, or one crude snake venom ( Crotalus horridus horridus). Snake, Gila monster and bee venoms exhibit higher PLB activity at an alkaline pH, while hornet venom PLB activity is greater at a neutral pH. Phospholipase A 2 (PLA 2) activity was determined using phosphatidylcholine and egg yolk as substrates. In reptile and bee venoms PLA 2 activity is much higher than PLB activity. In contrast, hornet venom PLA 2 and PLB activities are nearly equal. In reptile venoms PLA 2 activity is optimal at neutral pH, in contrast to PLB activity, suggesting that separate proteins or active sites are responsible for PLA 2 and PLB activities. The effect of boiling or heating venoms in either an acidic or alkaline milieu upon subsequent PLA 2 and PLB activity, measured at 37°C, was examined. The PLA 2 and PLB activities of all venoms tested were lost upon boiling at pH 9.4, except for the PLA 2 activity of Notechis scutatus venom which retained about 30% of its activity. Boiling at pH 5.5 results in greatly varying extents of retention of PLA 2 and PLB activities, dependent upon the venom examined. Therefore, the heat stability characteristics for each venom must be experimentally determined, not assumed. Boiling destroys PLA 2 and PLB activities of oriental hornet venom at about the same rate. No conclusive results were obtained from the heating studies as to whether PLA 2 and PLB activities reside upon the same molecule. However, PLA 2 and PLB activities of oriental hornet venom were separated using triple tandem column gel permeation chromatography, demonstrating the existence of two separate proteins for these activities.

Phospholipases A and B activities of the oriental hornet ( Vespa Orientalis) venom and venom apparatus

Phospholipases A and B activities of the oriental hornet ( Vespa orientalis) venom and venom apparatus. Toxicon 15, 141–156, 1977.—Oriental hornet venom is a rich source of both phospholipase A (PhA) and phospholipase B (PhB) activities. This was shown by incubating venom with egg yolk or with pure lecithin and lysolecithin. Activity was measured by titrating liberated fatty acids and by phosphorus analyses of separated phospholipids from thin-layer chromatographic plates. Both lecithin and lysolecithin were rapidly hydrolyzed by venom at pH 4 and 8. With egg yolk as substrate, the optimum Ph activity was observed at pH 5 although considerable activity was observed from pH 3·5 to 9·5. In contrast purified substrates showed greater activity at an alkaline pH, whether assay was in the presence of collidine-acetate or Tris buffer or in the presence or absence of ether. Ether dramatically changed the optimal pH for Ph activity, with egg yolk as substrate, from acidic to alkaline. It is not known whether these PhA and PhB activities are dual activities of a single enzyme or activities of two separate enzymes. The venom has neither PhC nor lipase activities. PhA and PhB activities were observed not only in pure venom (V), but also in venom sacs (VS) where the venom is stored, in the acid (venom) glands (H +) where the venom is produced, and in the alkaline (Dufours) gland (OH −) whose function is unknown. The release of free fatty acids from egg yolk at pH 5 was in the ratios of 1 (V): 0·13 (VS): 0·04 (H +): 0·30 (OH −). Measurements of Ph activities in combinations of the above preparations showed that strong activators or inhibitors of the enzyme are not present. The mid-gut, fat body and hemolymph of the hornet showed Ph activity only equal to about one-half of one per cent of that of the OH − gland. Antisera produced against V, VS, H + and OH − were cross-reactive and inhibited to varying degrees the Ph activities of each of the above preparations. The highest titre antisera were produced in rabbits injected with V or VS, in contrast to the low titres produced with H + and OH − glands. Low Ph activity is detectable in the venom sacs 2 days prior to emergence, with 20-fold higher activities being observed at 5 days of age. The oriental hornet may be extremely useful as a rich source of not only PhA but also PhB activities. The effects of this latter enzyme on biological systems have not been thoroughly evaluated. The drastic disruption of phospholipid structure and hydrophobic binding forces between phospholipid and protein, by the combined PhA plus PhB activities, may be responsible for some of the pharmacological actions of hornet venom.