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Biomedical subjects

A Klip

Publications and source records attributed to A Klip.

190 records · Page 11Linked to original sources
PubMed1979

Partial purification of the sugar carrier of intestinal brush border membranes. Enrichment of the phlorizin-binding component by selective extractions.

The [3H] phlorizin-binding component of brush border vesicles was enriched in situ by negative purification. Several procedures, known to effect selective solubilization of membrane components, were used separately or in combination to remove proteins unrelated to the binding. Deoxycholate ruptured the vesicles and released 67% of their protein, thereby increasing the specific [3H] phlorizin-binding activity of the pellet three-to fourfold. Extracting the deoxycholate-pellets with either NaI or alkaline solutions released up to 38% of the deoxycholate-insoluble protein without significantly affecting phlorizin binding. The polypeptide composition of the membranes at the different stages was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. A number of polypeptides present in the original vesicles could be ruled out as essential components of the [3H] phlorizin binding entity. Intact and deoxycholate-treated vesicles were subjected to proteolytic attack. Papain liberated sucrase and isomaltase from intact vesicles, but affected neither other Coomassie-stained bands nor phlorizin binding. Neither the protein composition nor the binding properties of sealed vesicles were influenced by trypsin or chymotrypsin. However, all the proteolytic enzymes tested on deoxycholate-treated membranes substantially reduced [3H] phlorizin binding and produced concomitantly the disappearance of several bands from the electrophoretic profile. Pretreatment of vesicles with papain, followed by deoxycholate extraction and incubation in alkaline media, increased the specific binding activity of the membranes up to ninefold by removing close to 90% of the protein. A limited number of polypeptides are suggested as possible candidates for the glycoside-binding site of intestinal brush borders.

Animals↗
PubMed1979

Distribution of sulfhydryl groups in intestinal brush border membranes. Localization of side-chains essential for glucose transport and phlorizin binding.

1. Brush border membrane vesicles from rabbit small intestine were found to contain 46 nmol SH groups/mg protein, 52% of which could react with 4,4'-dithiodipyridine, a membrane permeating probe. Only 18% of the total SH-groups reacted with the impermeant probe 5,5'-dithiobis(2-nitrobenzoic acid), indicating that only this fraction is externally located. 2. Brush border membrane vesicles could be disrupted by a gentle treatment with deoxycholate, releasing most of their electron-dense core material. In deoxycholate-treated vesicles most of the SH groups that reacted with 4,4'-dithiodipyridine react with 5,5'-dibiobis(2-nitrobenzoic acid), suggesting that both membrane surfaces became exposed to the extravesicular medium. 3. In intact vesicles (1.2 mg protein/ml), the binding of phlorizin (a competitive inhibitor of the monosaccharide transport system) was 50% inhibited by 67 microM of the penetrating organomercurial p-chloromercuribenzoate, but was about ten times less sensitive to the poorly permeating p-chloromercuriphenylsulfonate. In contrast, binding of phlorizin to leaky (deoxycholate-treated) membranes was equally susceptible to either reagent. 4. Mercurial inhibition of phlorizin binding could be reversed by dithioerythritol in both sealed and leaky membranes, whereas the less permeant thiol L-glutathione (reduced form) could only revert the inhibition in leaky membranes.

Animals↗
PubMedDate not supplied

Stimulation of glucose and amino acid transport and activation of the insulin signaling pathways by insulin lispro in L6 skeletal muscle cells.

The monomeric insulin analogue insulin lispro (Lys B28, Pro B29) is a rapid-acting insulin with a shorter duration of activity than human regular insulin. This compound has the advantage of reducing early postprandial hyperglycemia and the accompanying late hypoglycemia, thereby improving overall blood glucose control. To date, all published studies of the functional properties of insulin lispro have been conducted in whole animals. This study aimed to characterize the cellular actions of insulin lispro and the signals it elicits in an insulin-sensitive muscle cell line, L6 cells. Comparing the cellular actions of insulin lispro with those of human regular insulin, a number of observations were made. (1) Insulin lispro stimulated glucose and amino acid transport into L6 myotubes with a dose dependency and time course virtually identical to those of human regular insulin. (2) Insulin lispro was as effective as human regular insulin in stimulating time-dependent phosphorylation of insulin receptor substrate 1 (IRS-1), p70 ribosomal S6 kinase, and two isoforms of mitogen-activated protein kinase (ERK1 and ERK2). (3) Insulin lispro's ability to induce the association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase was similar to that of human regular insulin. (4) As with human regular insulin, 100 nmol of the fungal metabolite wortmannin completely inhibited insulin lispro stimulation of glucose uptake. We concluded that the cellular actions of insulin lispro are similar to those of human regular insulin with respect to glucose and amino acid uptake and that the biochemical signals elicited are also comparable.

Amino Acids↗
PubMedDate not supplied

Subcellular distribution and immunocytochemical localization of Na,K-ATPase subunit isoforms in human skeletal muscle.

The expression of Na,K-ATPase isoforms was investigated in human skeletal muscle membranes isolated by subcellular fractionation. The alpha 1, alpha 2, alpha 3 and beta 1 subunits were detectable in membranes prepared from the human soleus muscle. The alpha 1 subunit was largely detected in a fraction enriched with plasma membranes (PM), its abundance in an intracellular membrane fraction (IM) accounted for only 4% of that in the PM fraction. No alpha 1 subunits were detected in membranes of sarcoplasmic reticulum (SR) origin. The PM and IM fractions were enriched with alpha 2 subunits which were less abundant in the SR-enriched fraction. The abundance of alpha 2 molecules within the IM fraction was about 75% of that in the PM fraction when the total protein content for the two fractions was taken into account. Immunocytochemical studies confirmed the localization of the alpha 1 subunit to the muscle cell surface. The alpha 2 subunit was also found to be present in the cell surface but the observation that alpha 2 immunofluorescence was diffusely dispersed throughout the muscle fibre indicated that it was also present intracellularly, consistent with its biochemical localization in the PM and IM membrane fractions. The alpha 3 subunit was detected largely in the PM fraction but the lack of good antibodies to this isoform precluded an analysis of its immunocytochemical localization. The beta 1 subunit was enriched in the PM fraction but was also detected to a modest extent in the IM.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗