Jens Carlsen

Characterization of a second peptide with adipokinetic and red pigment-concentrating activity from the locust corpora cardiaca

Abstract A new peptide with both adipokinetic activity in the locust and red pigment-concentrating activity in the shrimp, can be readily separated from the adipokinetic hormone in extracts of corpora cardiaca (CC) from Schistocerca americana gregaria by the use of gel chromatography and the Leander adspersus bioassay. The new peptide accounts for 20% of the total biological activity in locust CC, and it is located principally in the CC glandular lobe. The amino acid composition of the pure peptide is: Asp, Thr, Ser, Glu, Gly, Leu, Phe, Trp. This composition is similar to, but not identical with the red pigment-concentrating hormone of the shrimp.

Microvillus membrane vesicles from pig small intestine Purity and lipid composition

Microvillus membrane vesicles from pig small intestine were isolated by a method based on hypotonic lysis, Mg2+-aggregation of contaminants and differential centrifugation. The purity of the membrane vesicles were established by measuring the activity of marker enzymes and the RNA and DNA content. The membranes were found free of contamination by other subcellular membrane fragments, except for a minor contamination with basolateral plasma membranes. The lipid composition was established and, based on weight percentage, the membrane contained neutral lipids, phospholipids, neutral glycolipids and gangliosides in the weight ratio of 18 : 50 : 29 : 2%. The amount of individual phospholipids and glycolipids were quantitated. Phosphatidylethanolamine, -choline, -serine, -inositol and sphingomyelin made up 17, 17, 6, 5 and 5%, respectively of the total lipid. The major glycolipids were two monohexosylceramides containing glucose and galactose as the carbohydrate component, a dihexosylceramide containing galactose as the only carbohydrate component and two pentahexosylceramides containing fucose, galactose, glucose and hexosamine (either N-acetylglucosamine or N-acetylgalactosamine) in the molar ratio of 1 : 2 : 1 : 1.

Purification and chemical structure of the red pigment-concentrating hormone of the prawn Leander adspersus.

The red pigment-concentrating hormone of the eyestalks of the prawn Leander adspersus has been purified. It is an octapeptide with the following amino acid composition: Asp1, Glu1, Gly1, Leu1, Phe1, Pro1, Ser1, Trp1. The complete identity in purification behavior, in amino acid composition, in electrophoresis, and in partition chromatography with the previously purified red pigment-concentrating hormone from the prawn Pandalus borealis, strongly indicate structural identity between the hormones of the two species.

Insulin Stimulated Glycogen Synthesis in Isolated Rat Hepatocytes: Effect of Protein Kinase Inhibitors

Glycogen synthesis was studied in rat hepatocytes isolated by EDTA perfusion. Insulin induced a one and a half to twofold increase in glucose incorporation into glycogen. Insulin stimulated glycogen synthesis was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin (IC50 approximately 40 nM) and LY 294002 (IC50 approximately 20 microM) and the mitogen-activated protein kinase kinase inhibitor PD 98059 (IC50 approximately 40 microM). Wortmannin was without appreciable effect on non-insulin stimulated glycogen synthesis, while LY 294002 and PD 98059 also inhibited the non-insulin stimulated glycogen synthesis. Rapamycin, an inhibitor of p70 ribosomal protein-S6 kinase, was without effect on glycogen synthesis regardless of insulin stimulation.

EVIDENCE FOR AN ADIPOKINETIC FUNCTION OF THE RPCH ACTIVITY PRESENT IN THE DESERT LOCUST NEUROENDOCRINE SYSTEM

1. Red pigment concentrating hormone-like activity (RPCA) has been demonstrated and quantified in the head or cephalic neuroendocrine system of several insects. Schistocerca gregaria adults contained the highest levels of RPCA.2. RPCA was found in all post-embryonic life cycle stages of S. gregaria, and seems to be present in equal quantities in both males and females. Adult locusts contain significantly more RPCA than do immature animals.3. RPCA is concentrated in the locust CC glandular lobe hut is also present in the CC storage lobe, brain and hemolymph of both mature and immature animals. Significant variations in CC RPCA content have been demonstrated in both mature and immature stages.4. Synthetic red pigment concentrating hormone (RPCH) is adipokinetic in both intact and neck-ligatured S. gregaria adults and in intact immature animals. Doses of about 6 ng and 50 ng, respectively, result in minimal and maximal adipokinetic responses. RPCH selectively elevates hemolymph 1.2-diglycerides.5. RPCH in la...

Multiple Forms Of Soluble Butyrylcholinesterase In Human Brain

Summary 1. 30-55% of the butyrylcholinesterase (EC 3.1.1.8) activity of human brain was present in an aqueous extract. Particle-bound butyrylcholinesterase could not be made soluble. 2. In DEAE-cellulose chromatography soluble butyrylcholinesterase was separated in one inhomogeneous main fraction and several minor fractions. Gel chromatography on Sephadex G-200 resolved the main fraction into two inhomogeneous fractions, one of higher molecular weight (300 000-400 000), the other of lower molecular weight (50 000-150 000). 3. Electrofocusing of the butyrylcholinesterase fraction of higher molecular weight revealed two inhomogeneous fractions in the pH ranges 3.8-4.7 and 5.6-8.2. By treatment with neuraminidase (EC 3.2.1.18) the most acid subfractions disappeared and alkaline subfractions appeared. The acid and the most alkaline subfractions may originate from plasma trapped in the tissue. Electrofocusing of the fraction of lower molecular weight showed at least 6 subfractions in the pH range 5.6-8.0. Treatment with neuraminidase had no effect on their isoelectric points. 4. Brain tissue thus contains at least 4-5 soluble butyrylcholinesterase fractions of high molecular weight with isoelectric points ranging from 5.6 to 7.0 and at least 6 low-molecular-weight fractions with isoelectric points from 5.6 to 8.0.

Membrane Topology of Insulin Receptors Reconstituted into Lipid Vesicles

Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation was confirmed by immunogold labeling of the α- and the β-subunit of the receptor. Immunogold labeling of the C-terminus of the β-subunit indicates that it resides about 6 nm off the membrane, while two α-subunit epitopes were labeled at about twice this distance, confirming that the α-subunit is harbored in the cross-bar of the T-structure.

Reconstitution of a protein into lipid vesicles using natural detergents

A method is described for reconstitution of a protein into lipid vesicles using one of the natural detergents lysophosphatidylcholine or lysophosphatidic acid. The intestinal microvillus enzyme, aminopeptidase N (EC 3.4.11.2) is incorporated into lipid vesicles prepared from a total lipid extract of the microvillus membrane. The method is based on fusion of aminopeptidase-lysophospholipid micelles with liposomes prepared by sonication. The incorporation of the protein into the lipid bilayer is analyzed by gel permeation chromatography and sucrose density gradient centrifugation. The coincidence of the protein and lipid profiles is used to evaluate protein incorporation. The incorporation is visualized by electron microscopy with negative staining. The method has the advantage of using natural detergents, lysophospholipids, which are minor but natural constituents of biological membranes. The method could be of value as a tool in studies of mechanisms of insertion of newly synthesized proteins into biological membranes.

Purification of microvillus membrane vesicles from pig small intestine by adsorption chromatography on sepharose

Abstract A simple, rapid method for the preparation of pure microvillus membrane vesicles from pig small intestine is described. The method is based on the ability of agarose beads to adsorb selectively the impurities, mainly basolateral membrane fragments, from a microvillus vesicle preparation isolated by hypotonic lysis, Mg2+ aggregation of contaminants and differential centrifugation.

Adipokinetic activity of shrimp and locust peptide hormones in butterflies

Abstract Synthetic shrimp red-pigment-concentrating hormone (RPCH) and pure locust adipokinetic hormone (AKH) both cause significant, dose-dependent elevations of hemolymph lipids in the Monarch butterfly, Danaus plexippus , and in the Painted Lady butterfly, Vanessa cardui . Both hormones are effective when administered in picomole amounts; AKH appears to be about 100 times more active than RPCH. These results suggest a highly conservative evolution of some mandibulate arthropod neurohormones.