Maria Gaczyńska

Proteolytic self-digestion of bovine erythrocyte membranes

Self-digestion of bovine erythrocyte membrane proteins was studied in isolated membrane preparations during prolonged incubation at 37 C. Protease activities associated with the membrane result in progressive degradation of all main erythrocyte membrane proteins, in particular spectrin and Band 3, and formation of lower molecular weight products which have been tentatively assigned to parent molecules. Membrane protein self-digestion occurs in a broad pH range (2-11), is inhibited by increased ionic strength and by inhibitors of metalloproteases, cysteine and serine proteases, and activated by low concentrations of SDS. Self-digestion also takes place in NaOH-stripped erythrocyte membranes. The activity of a protease involved in the self-digestion, of apparent molecular weight of about 35,000, was renatured after SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins.

Hyperthermia, unlike ionizing radiation and chemical oxidative stress, does not stimulate proteolysis in erythrocytes

1. Oxidative stress by phenazine methosulfate stimulated proteolysis in erythrocytes. 2. Gamma-irradiation of erythrocytes in the range of 50-1000 Gy also resulted in the induction of proteolysis. 3. Though it has been suggested that hyperthermia imposes an oxidative stress on a cell, hyperthermic exposure of erythrocytes (30 min, 39-49 degrees C) did not stimulate proteolysis during subsequent incubation of whole cells or hemolysates. 4. Proteolytic degradation of spectrin was accelerated during incubation of membranes isolated from cells heated above 45 degrees C but this effect seems to be due rather to thermal denaturation of spectrin than to oxidative modification of cellular proteins by hyperthermia.

Changes in the proteolytic activity of human erythrocyte membrane during red cell aging

The action of endogenous membrane proteinases in membranes isolated from human red cells of various ages was assayed by three groups of methods: (1) determination of the amount of protein fragments released to the acid-soluble fraction; (2) monitoring of changes in ESR spectra of maleimide spin-labeled erythrocyte membranes; (3) electrophoretic methods: a two-dimensional analysis and analysis of the activity inside SDS-PAGE gels. For all the methods the effects of proteinase action were highest in ghosts isolated from the erythrocytes of middle age.

Abnormal degradation of erythrocyte membrane proteins in hereditary spherocytosis

The degradation rate of erythrocyte membrane proteins by membrane-bound proteases was compared in healthy controls and in patients with hereditary spherocytosis (HS). An increased degradation rate of spectrin and a decreased digestion rate of Band 3 were found in HS patients. These differences may be due to altered accessibilities of membrane proteins to proteases and/or to changes in the pattern of membrane-associated proteases and may be connected with the decrease in the spectrin: Band 3 ratio reported for erythrocyte membranes in HS.

Crosslinking of membrane proteins during erythrocyte ageing

Membrane proteins of bovine erythrocytes were crosslinked with cupric di(1,10-phenanthroline) and analysed by one-dimensional and two-dimensional SDS-polyacrylamide gel electrophoresis. An increase in crosslinking of the Band 3 protein and of spectrin was found with increasing erythrocyte age suggesting an increased aggregation of main membrane proteins in aged erythrocytes.

Proteolytic activity of human erythrocyte membrane during ghosts preparation

1. Proteins in human erythrocyte membranes after red blood cells hemolysis revealed relatively high rate of self-digestion. 2. This indicates hemolysis as a critical moment for membrane proteases activation. 3. The detailed pattern of band 3 protein and spectrin degradation during ghosts preparation was more complicated and reflected both the changes in proteolytic susceptibility and extraction of some proteases. 4. Further extraction of membrane proteins by alkali stripping resulted in an increase in the self-digestion rate and decrease in the degradation rate of an exogenous substrate.

Endogenous proteolysis of the human erythrocyte membrane as studied by two-dimensional gel electrophoresis and electron spin resonance

1. Endogenous proteolysis in human erythrocyte membranes was studied in human erythrocyte membranes incubated at 37 degrees C by monitoring changes in 2-D electrophoretic pattern of membrane polypeptides and in the spectra of maleimide-spin labeled membranes. 2. A strong effect of exogenous proteases derived from contaminating other blood elements was found, resulting in formation of specific spots on 2-D electropherograms, requiring very careful leukocyte removal in investigations of red cell membrane protein composition and proteolysis. 3. Studies of the effects of protease inhibitors and Ca2+ confirmed a complex pattern of endogenous red cell membrane proteolysis (self-digestion) involving many substrates and enzymes. 4. A promoting effect of high concentrations (150 mM) of Ca2+ on endogenous red cell membrane proteolysis was found.

Proteolytic susceptibility of membrane proteins during erythrocyte aging

Susceptibility of membrane proteins to digestion with chymotrypsin was compared in various age fractions of bovine erythrocytes by SDS-polyacrylamide gel electrophoresis. The extent of protein digestion was found to be higher in membranes of older erythrocytes, as judged from the disappearance of original protein bands and accumulation of digestion products. These results are consistent with the hypothesis of a proteolytic generation of a senescent cell antigen in red blood cells.

Proteolytic activity in electropherograms (SDS-PAGE) of bovine erythrocyte ghosts

1. Activity of proteases, strongly related with erythrocyte membrane, was analysed employing a new methodological approach. 2. Intact bovine ghosts, ghosts depleted in peripheric proteins or purified Triton X-100 and ghost extracts were electrophoresed and the proteolytic activity in the gel fragments (SDS-PAGE) was assayed. 3. At least two proteases that were inhibited by EDTA and PMSF were found.

A spin-label study of membrane proteins and internal microviscosity of erythrocytes in hereditary spherocytosis

Electron spin resonance (ESR) spectra of erythrocyte membranes of patients with hereditary spherocytosis (HS) and of healthy controls labeled with a maleimide spin label did not differ significantly both before and after prolonged incubation at 37 degrees C. It suggests that the different behavior of spin-labeled HS erythrocyte membranes upon incubation at a higher temperature reported previously is due indeed to structural abnormalities of HS red cell membranes and not to alterations in their proteolytic activity. Measurements of the rotational correlation time of Tempamine spin probe demonstrated a significant elevation of internal microviscosity of erythrocytes in HS, more pronounced in non-splenectomized patients.