Jakob Donnér

Lipase-colipase interactions during gel filtration. High and low affinity binding situations.

The interaction of porcine pancreatic lipase and colipase was studied during gel filtration in columns eluted with a variety of buffers. High and low affinity binding situations were observed under different conditions. Low affinity binding could only be detected at the high lipase-colipase concentrations encountered during batch purification (10(-3)-10(-4) M). Even in this situation the rapid dissociation of the weak complex during filtration resulted in considerable separation of the two proteins. High affinity binding of lipase to colipase was observed at protein eluant concentrations as low as 10(-8) M on columns equilibrated with oleic acid-taurodeoxycholate mixed micelles. This binding did not take place on columns equilibrated with simple bile salt and mixed phosphatidylcholine-cholesterol-bile salt micelles. Colipase alone exhibited strong binding to phosphatidylcholine and fatty acid mixed bile salt micelles when applied together in a sample on columns eluted with pure bile salt micelles, lipase did not. The relevance of the high affinity complex to the lipase . colipase . substrate complex is discussed.

The polar interactions between pancreatic lipase, colipase and the triglyceride substrate.

Pancreatic lipase is an enzyme that uniquely interacts with the interface of water-insoluble ester substrates but also with hydrophobic interfaces in general [l] . These hydrophobic interactions lead to the rapid irreversible denaturation of the enzyme and cannot be considered to be of interest under physiological conditions. In the in vivo situation in the small intestinal contents lipase is functioning in the presence of bile salts which by their general detergent effect desorb lipase from the substrate interface by braking or preventing hydrophobic interactions [2]. Under certain conditions of pH and salt, lipase can bind to the substrate and be catalytically active also in the presence of bile salt, indicating that polar interactions can be important [2,3]. Colipase, a polypeptide cofactor of pancreatic juice, binds to lipase substrate in bile salt solution and serves as an anchor for lipase to the substrate [4-61, thus enabling lipase to be active in bile salt solution over a wide pH range. It is the objective of the present study to demonstrate that ‘polar’ rather than hydrophobic bondings are important for the attachment of lipase and colipase to the bile salt laden substrate interface and thus for the biological function of the enzyme. Evidence is also presented to show the specific binding of these two proteins most probably by hydrogen bonding to the carbonyl groups of the substrate.

On the binding of bile salt to pancreatic lipase.

The binding of taurodeoxycholate to pancreatic lipase and a few other proteins has been studied with equilibrium dialysis and in gel filtration experiments. A three compartment dialysis cell has been used; with this cell, complete equilibration is not necessary for calculation of the binding even at bile salt concentrations above the critical micellar concentration. The results indicate that taurodeoxycholate does not bind to lipase below the critical micellar concentration, that the binding starts in the critical micellar concentration range of the bile salt and reaches around 12 mol taurodeoxycholate per mol of lipase at taurodeoxycholate concentrations well above the critical micellar concentration. Previous results indicating a binding of maximally 1-2 mol taurodeoxycholate/mol lipase were too low, depending on the experimental conditions in which complete equilibration was not obtained. The binding isotherm for taurodeoxycholate to lipase is similar to that for co-lipase; colipase and lipase in mixture bind as much taurodeoxycholate as the sum for the single proteins. Taurodeoxycholate binds to ribonuclease and chymotrypsinogen to a similar extent as to lipase.

Medical education in Sweden

Undergraduate medical education in Sweden has moved from nationally regulated, subject-based courses to programmes integrated either around organ systems or physiological and patho-physiological processes, or organised around basic medical science in conjunction with clinical specialities, with individual profiles at the seven medical schools. The national regulations are restricted to overall academic and professional outcomes. The 5½ year long university undergraduate curriculum is followed by a mandatory 18 months internship, delivered by the County Councils. While quality control and accreditation for the university curriculum is provided by the Swedish National Agency for Higher Education, no such formal control exists for the internship; undergraduate medical education is therefore in conflict with EU directives from 2005. The Government is expected to move towards 6 years long university undergraduate programmes, leading to licence, which will facilitate international mobility of both Swedish and foreign medical students and doctors. Ongoing academic development of undergraduate education is strengthened by the Bologna process. It includes outcome (competence)-based curricula, university Masters level complying with international standards, progression of competence throughout the curriculum, student directed learning, active participation and roles in practical clinical education and a national assessment model to assure professional competence. In the near future, the dimensioning of Swedish undergraduate education is likely to be decided more by international demands and aspects of quality than by national demands for doctors.

Growth hormone increases phosphoinositide turnover in rat adipocytes that are sensitive to the insulin-like action of the hormone

SummaryThe effect of pituitary human growth hormone (hGH) on the 32P-labelling of phosphoinositides and phosphatidic acid was studied in noradrenaline-stimulated rat adipocytes which were either responsive or non-responsive to the antilipolytic (insulin-like) effect of hGH. In cells responsive to the insulin-like effect of hGH, hormone treatment resulted in a marked increase of the 32P-labelling of phosphatidic acid and phosphatidyl inositol in the plasma membrane, high density microsomal, and low density microsomal fractions. The increased 32P-labelling most likely reflects an activation of phosphoinositide phospholipase C.

The acute GH action in rat adipocytes is associated with enhanced phosphorylation of a 46 kDa plasma membrane protein enriched by GH-Sepharose

The acute anti‐lipolytic effect of human growth hormone (hGH) in maximally noradrenaline‐stimulated intact rat adipocytes was selectively associated with increased phosphorylation of a 46 kDa plasma membrane protein which was highly enriched by hGH‐Sepharose chromatography. The same protein was also phosphorylated by an endogenous protein kinase in isolated plasma membranes, although then no hGH effect could be demonstrated. About 14% of the phosphate incorporated into the protein in isolated plasma membranes was found in tyrosine residues and the remainder in serine and threonine. The possible relation of the 46 kDa protein with the hGH plasma membrane receptor is discussed.