Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A2 with micellar lipid/water interfaces of competitive inhibitors.

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.     

p82H identifies sequences at every human centromere

Summary A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.     

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.     

Effects of gum arabic on lipase interfacial binding and activity.

We investigated the surface behavior of gum Arabic (GA) as well as its effects on the lipolytic activity of human pancreatic lipase (HPL) and Humicola lanuginosa lipase (HLL), using emulsions of triacylglycerols (TAG) with various chain lengths. The effects of GA on the interfacial binding of HPL were also investigated. In the presence of 4 mM sodium taurodeoxycholate (NaTDC), GA (3% w/v, final concentration) had no effect on the HPL activity measured in the presence of colipase, whatever the type of TAG used. However, in the absence of bile salts or at low bile salt concentrations, GA inhibited the HPL activity when trioctanoin (TC8) and purified soybean oil (PSO) were used as substrates. At 3% (w/v, final concentration), GA strongly desorbed pure HPL from the TC8 interface and the classical anchoring effect of colipase was clearly observed. Both crude and dialyzed GA solutions were found to be highly tensioactive at the air-water as well as the oil-water interface using the drop technique. In conclusion, GA, or a putative contaminant present in GA, was found to be surface active and to have similar effects to those of bile salts on the interfacial binding and activity of HPL.     

Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.