Solubilization of Certain Proteins from the Human Erythrocyte Stroma<sup>*</sup>

Mitchell, Carolyn D.; Hanahan, Donald J.

doi:10.1021/bi00865a008

Cited by 107 publications

(27 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Stroma prepared with added MgCI, was treated with 1.2 M NaCl solution as described by Mitchell and Hanahan [7]. Aliquots of the supernatant solutions were dialyzed a t 4 "C against distilled water and lyophilized; lipids were extracted from the lyophilized residues.…”

Section: Solubilization With Hypertonic Salinementioning

confidence: 99%

“…Investigations of Dodge et al [3] and Mitchell et al [7] have shown that human erythrocytes do not yield intact "ghosts" unless certain precautions are taken during the hemolysis procedure. Hemolysis of human erythrocytes in hypotonic buffers of 20-25 mosM and pH > 7.0 and subsequent washings yield ghosts that are essentially free of hemoglobin and retain virtually all of their membrane constituents, including acetylcholinesterase activity.…”

Section: Effect Of Magnesium Ionsmentioning

confidence: 99%

“…It has been shown by Mitchell and Hanahan [7] that treatment of human stroma with hypertonic NaCl solutions affects a partial solubilization of stromal lipids, proteins and acetylcholinesterase without complete disruption of the stromal structure. Burger et al [4] extended this procedure to intact bovine erythrocytes.…”

Section: Effects Of Hypertonic Salinementioning

confidence: 99%

“…This would be consistent with the observation of Burger et al [4] that the membrane fragment released in the absence of Mg2+ during hemolysis of bovine erythrocytes behaves as a lipoprotein. They suggest that the acetylcholinesterase of the bovine erythrocyte membrane outer surface may exist as a lipoprotein complex all the more since an acetylcholinesterase-lipoprotein preparation could be obtained from human stroma [7].…”

Section: 'mentioning

confidence: 99%

See 3 more Smart Citations

Studies on the Attachment of the Bovine J Blood‐Group Substance at the Erythrocyte Membrane

Schröffel¹,

Thiele²,

Koch³

1971

European Journal of Biochemistry

View full text Add to dashboard Cite

By means of quantitative studies of the J activity of bovine intact erythrocytes and of erythrocyte lipids it is concluded that all of the J activity of J-containing cells is due t o a lipid and that all J substance is present on the erythrocyte surface and thus available for J antibody. No J substance seems to be buried in the depth of the membrane.During the process of hemolysis and subsequent washings bovine erythrocytes release a considerable portion of their membrane constituents in a "soluble" form. I n order to prevent a disruption of ghosts a small amount of MgCl, must be added to the hemolyzing mixture. We observed a loss of more than 30°/, of the original J activity along with about 150/, of various stroma constituents unless MgCl, had been added to the hemolyzing mixture.By treatment of bovine stroma (prepared in the presence of added MgC1,) with organic solvents of increasing polarity two lipid fractions, "loosely" and "strongly" bound lipids, can be obtained. The J activity was found only in the "strongly" bound fraction along with the majority of glycolipids.By treatment of bovine J-cell ghosts with hypertonic saline part of the stroma constituents, including the J substance, is solubilized. It is concluded that the J substance, though secondarily absorbed from the serum onto the erythrocyte surface, is fully integrated to the other membrane constituents.The bovine J blood-group substance is primarily dissolved in the blood plasma [l] and is absorbed from there onto the erythrocytes during a postnatal period. Previous reports from these laboratories [2]have shown that the J substance of serum is a lipoprotein and that its determinant is a glycosphingolipid. The goal of further studies has been to investigate how the J substance is attcahed to the erythrocyte membrane. This paper described the &st results of such investigations. Some of the following results have already been presented at the XIIthInternational Conference on Animal Blood Groups (Budapest, July 1970). MATERIALS AND METHODSBlood was drawn from cattle which were classed as being with or without the J substance. As the anticoagulant one part by volume of a solution containing 2 0 g sodium citrate and 5 g sodium chloride per 1000 ml water was used for 4 parts of blood.I n order to obtain erythrocytes containing J substance for the hemolysis-inhibition tests one part by volume ofa solution containing 32 g sodium citrate,Enzyme. Acetylcholinesterase (EC 3.1.1.7). 10 g glucose, 5 g sulfanilamide, 0.04 g RivanolQ and 0.2g sodium cyanide per 1000ml water was used for about 4 parts of blood. This mixture was stored a t 4 "C for up to 6 weeks. From this mixture small samples of erythrocyte suspensions were freshly prepared by centrifugation and sufficient washing with an excess of isotonic (0.15M) saline a t room temperature.Serum was obtained from freshly drawn blood by allowing it to stand a t room temperature overnight.For serological tests anti-J serum was used which had been checked in international comparison tests. I n addition, an anti...

show abstract

Section: Solubilization With Hypertonic Salinementioning

confidence: 99%

Section: Effect Of Magnesium Ionsmentioning

confidence: 99%

Section: Effects Of Hypertonic Salinementioning

confidence: 99%

Section: 'mentioning

confidence: 99%

See 2 more Smart Citations

Studies on the Attachment of the Bovine J Blood‐Group Substance at the Erythrocyte Membrane

Schröffel¹,

Thiele²,

Koch³

1971

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…There is evidence that this soluble material may be associated with lipid or other protein [ 1,4,5] . Acetylcholinesterase has also been solubilised as a lipoprotein by washing erythrocytes with 1.2 M sodium chloride [6]. Accordingly, the effect of phospholipase A on the acetylcholinesterase solubilised by Triton Xl00 treatment of erythrocyte membranes has been investigated.…”

Section: Introduction the Effect Of Phospholipase A On Human Erythrocmentioning

confidence: 99%