Brush border lactase-phlorizin hydrolase carries two catalytic sites. In the human enzyme lactase comprises Glu-1749, phlorizin hydrolase Glu-1273. The proteolytic processing of pro-lactase-phlorizin hydrolase by (rat) enterocytes stops two amino acid residues short of the N-terminus of`mature' final, brush border lactase-phlorizin hydrolase. Only these two amino acid residues are removed by luminal pancreatic protease(s), probably trypsin.z 1998 Federation of European Biochemical Societies.

The solubilization of plasma and organelle membranes by diheptanoylphosphatidylcholine (DHPC) has been studied. This short-chain phosphatidylcholine is shown to act as a mild detergent, solubilizing effectively both kinds of membranes at DHPC concentrations of 10-20 mM (0.5-1%). The size of the resulting mixed protein-lipid-DHPC micelles ranges between 5 and 8 nm. The protein conformation and hence the enzymatic activity are well preserved over a rather large DHPC concentration range (up to 4-5 times the DHPC concentration required for solubilizing the membranes). Evidence is presented that short-chain phosphatidylcholines are superior to most detergents commonly used by biochemists. This is true not only regarding its excellent dispersing power on both phospholipid bilayers (Gabriel & Roberts, 1986) and biological membranes but also as to its capacity to preserve the native protein structure and hence enzymatic activity in the solubilized state. Due to its special properties DHPC lends itself very well not only to membrane solubilization but also to the purification of the solubilized membrane proteins and reconstitution of the proteins into simple lipid bilayers. Concerning the mechanism of membrane solubilization, evidence indicates that DHPC interacts primarily with the lipid bilayer of the membrane and not with the membrane proteins. DHPC solubilizes membranes by being distributed into the lipid bilayer and breaking it up. In the resulting small mixed micelles, the protein remains associated with its preferred intrinsic membrane lipids and is thus stabilized. The protein-intrinsic lipid complex is successfully shielded from unfavorable contacts with H2O by DHPC-intrinsic lipid interactions.

Calcium has been demonstrated to play an important role in hepatocyte damage during ischemia/reperfusion phases. Calcium influx was determined in primary cultured rat hepatocytes submitted to a succession of warm hypoxia and reoxygenation phases in the presence of diltiazem, gallopamil and a Na⁺/H⁺ antiport inhibitor, HOE-694. Only diltiazem significantly inhibited calcium influx with higher potency after reoxygenation than after hypoxia only, suggesting a complex mechanism of action of diltiazem which could act on different physiological functions involved in Ca²⁺ invasion of hepatocytes after hypoxic insult.

To assess the role of pancreatic proteases in the proteolytic processing and in the postweaning decline of lactase-phlorizin hydrolase (LPH), we have determined lactase activity and the different LPH forms in postweaned rats in which a jejunal loop was excluded from contact with pancreatic secretions by a jejunal bypass procedure. As a control for the absence of pancreatic proteases, pro-sucrase-isomaltase (proSI), which is known to be split by pancreatic proteases into heterodimeric SI, was used. Nearly all proLPH was processed to mature LPH, indistinguishable from LPH isolated from control animals. SI was found only in the unsplit pro form, whereas it was normally processed to the heterodimeric SI in the control tissues. There were no significant differences in lactase and sucrase activities in operated and in sham-operated control animals. We conclude that pancreatic secretions are not essential for the processing of proLPH to LPH or in the postweaning decline of LPH.

The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zero trans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and delta psi = 0. Under zero trans condition and delta psi = 0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (Kt = 88 microM) and a low affinity for sodium (Kt = 57.7 mM) (site I), the second one with a low affinity for pyruvate (Kt = 6.1 mM) and a high affinity for sodium (Kt = 23.9 mM) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25 mM and 8 mM pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously. Under chemical equilibrium (delta psi congruent to 0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation was n = 1.7. The direct method of measuring Na+/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives a n = 1.67. Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).