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From www.bloodjournal.org by guest on July 31, 2017. For personal use only. Protease Activity in the Human to the Cell Localization By Samir Proteolytic activity erythrocyte was membrane. This be ascribed to the suspension. the soluble more active against IN the which against the could not extracted in cell subunits mole- experiments activity TWO CIRCUMSTANCES units of hemoglobin are but not activity of from dent on and ATP for the membrane-free was temperature an be mem- to O.75-M protease or than could cell by exposure time chains activity erythrocyte The system beta The from require confirmed proteolytic in degrading hemolysate. was hemoglobin Burka chains. branes. from and R. active alpha hemoglobin tetrameric membrane more cell absent Membrane Edward the leukocytes totally of and human to contaminating was Ballas mature activity. Pulse-chase that the localized portion than cules. in K. Erythrocyte: and KSCN. dependid activity. was hemoglobin or the alphaand beta-chain to attach to the human red cell membrane. known not energy-generating subFirst, as the red cell ages in the peripheral circulation, small but progressively amounts of hemoglobin become bound to the cell membrane.’ Second, in which there is unbalanced synthesis of the alpha and beta subunits increasing in conditions of hemoglo- bin, the instance In the molecules the chains produced it is not known membrane in excess what role, plays erythrocyte. in However, thalassemia syndromes, chains to the membrane cells.3 The manner membrane and senescence the in which the erythrocyte and the hemoglobins within the proteins. The findings indicate From the Medical Cardeza College. Foundation 26. 1978; accepted July by USPHS Presented San Diego. Address Philadelphia. © Blood. 1979 Vol. for Pa. Supported Grant in preliminary (‘alif. December. reprint requests to the erythrocyte of of protein human erythrocyte unknown. does subunits, Research, Department Proteolytic to the cell a part in possibility we hemoglobin contain and of to the of abnormal In order to investigate this human erythrocyte to degrade the the of excess hemoglobin destruction of red attachment remain first to circulating cells and have been localized that these enzymes might play Hemazologic January normal for degradation hemoglobin and hemoglobin to the red cell membrane. Philadelphia. Submitted that the is characteristic that attachment role in premature responsible hemoglobin chains.79 ability of the mature activity directed against this activity is confined of which reacts processes cell membrane.2’3 of hemoglobin death situation, enzymes are present in human erythroid membrane.56 It has been suggested destroying excess have studied the and second there is no question plays a prominent is not clear, normal the in also attach to the if any, attachment proteolytic virtually Medicine. all of Jefferson 9, 1979. AM-13431. form at the 20th Annual Meeting of the American Society of Hematology. 1977. to S. K. Ballas, M.D., Department of Medicine. Jefferson Medical College. Pa. /9107. by Grune & Stratton. 53. No. 5 (May), 1979) Inc. ISSN 0006-4971/79/5305-0008$Ol.0O/O 875 From www.bloodjournal.org by guest on July 31, 2017. For personal use only. 876 BALLAS MATERIALS Erythrocytes three the from times 310-mOsm in membranes hemolysate buffer, were in 10-12 7.4, until hemoglobin reticulocytes from hemoglobin subunits Enzyme assay membrane M), tube then pH 7.4, and g for in 10-20 frozen the amount result were also carried to the 20 and thawed and method mm. volumes of protein The washed of Dodge,’#{176} membrane-free of 20-mOsm three times phosphate and suspended was determined.” and a high preparing proportion of membrane-free form.’2 The alpha and beta on CM-Sephadex.’3 counter intact was determined 0.75-M ofAipha and preparation by column of Beta globin degraded The mercaptoethanol.’4 at intervals acid of the fragments added supernatant to was radioactivity.’2 in the assay of protease up to 24 were to acid-soluble assay to acid-soluble of Morrison cells, incubation mixture. activity so that and Neurath4 in a final Assays was designed denaturization the samples of Lowry.” The by incubating concentration for use as the enzyme of of 0.75 were centrifuged extract. The enzyme extract human M of potassium at 40,000 amount of protein was used in the assay g for 30 in the system used as a control. Hemoglobin by chromatography and I ml or 5 X the results. red blood KSCN hemolysate (approximately at 37#{176}C, and of substrate of enzyme. by the method by the method with chains centrifugation, amount substrate was aspirated membrane-free system incubated the protein not influence of human of 1 .5 ml of 20% tricholoracetic Following as a source I 8 hr at 0-4#{176}C.Following previously, water; to determine was extracted supernatant were of the original cells 2 ml or hemoglobin an energy-generating the tubes of radioactive activity contained hemoglobin protein. Activity and the clear Following cells containing described’2 to the carbonmonoxy 3 ml, containing as percentage or components Separation blood chromatography in 1 ml of ice-cold ofProtease for of substrate, would cells, supernatant mix the incubation proteolytic thiocyanate volume scintillation on degradation Extraction the hemoglobin by column undegraded out using during washed as previously .5 mg of substrate placed is expressed to depend by incubating [‘4C]leucine a final of a master were in a liquid The isolated were converting 0.5-1 to precipitate described membranes of the labeled aliquots red blood 40,000 The separated in 10 l addition protein at washed by centrifugation according System system, and Following counted centrifugation separated lysing were of after were suspension, hr 0.5-mi were After membranes buffer, presence them Assay The 7.4. the was obtained in the hemolysate mm, colorless. by METHODS blood pH BURKA of Substrate Labeled The and AND venous buffer, sedimented ml of Krebs-Ringer Preparation each phosphate was removed, pH l0’ 30 cc of heparinized AND the Chains acid-acetone on CM-52 method, in the presence the of 8-M alpha and beta subunits were urea.” RESULTS The bin initial within studies the red assay system freeze-thawed observed in hemoglobin the cell. containing either hemoglobin-free a classic in the membrane-free activity determined blood lysate human hemolysate in degrading substrate location Labeled normal red cell cell-free erythroid was present was that can degrade incubated in the hemoglodescribed human hemolysate or a suspension membranes. No proteolytic activity system,’2 cell tetrameric of activity substrate indicating is of extremely in the assay hemoglobin, that low system or alpha proteolysis activity. there and of was was beta of When only very little hemoglo- bin chains, during a period of 24 hr at 37#{176}C(Fig. 1). However, when the same substrates were incubated in the presence of a freeze-thawed membrane suspension, approximately 20% of the tetrameric hemoglobin was degraded during 24 hr, From www.bloodjournal.org by guest on July 31, 2017. For personal use only. ERYTHROCYTE MEMBRANE PROTEASE 877 ACTIVITY 0 w ID 4 HEMOGLOBIN E.1 LI 50 #{176}“ CHAIN Fig. 1 . globin and CHAIN ,9H Degradation hemoglobin of radioactive hemosubunits by erythro- cyte components. The proportion of radioactive I-. to acid-soluble the presence 25 bars indicate the substrate degraded fragments when of membrane-free incubated in hemolysate or hemoglobin-free membrane for 24 hr at 37”C. The amount of radioactive substrate in the assay system. whether tetrameric hemoglobin or hemoglobin chains. was 0.5 mg -.2 0 and there was even greater activity in degrading These findings indicate that the activity hemoglobin or its subunits is confined active against freezing hemoglobin and thawing, activity to the were incubated subunits. it was Since not possible inner or outer surface in the same assay hemoglobin the from of the system membranes these directly related the amount of membrane suspension of tetrameric hemoglobin degradation 2). Degradation to the amount had membrane. there was in the absence rate of substrate degradation Omission decrease alpha chains of membrane cell studies or alpha beta fragmented the in the assay was increased, chains also as noted of membrane, was also time-dependent against 20%-25% hemoglobin, greater the than by protease above, was system. the rate of degradation that of alpha chains. taken to ensure that the experiment was carried out under amount of substrate did not limit the rate of the reaction. The at 0#{176}C. did not against of Care 8 Li 0 Li 4 a: U) a’ a U) Fig. 2. Degradation bin and hemoglobin amounts of erythrocyte Conditions of incubation Fig. 1. of radioactive hemoglosubunits by increasing membrane susp#{149}nsion. were as described for beta was conditions in which the These findings suggest ID Li 0 4 a: of (Fig. minimal. and did not proceed As rate increased of AlP or the energy-generating system from the assay mixture enzyme activity. Figure 2 also illustrates that although the activity chains exceeds that was, on the average, 1). surface of the cell. or hemoglobin subunits system and been to localize suspension in the assay (Fig. to degrade native and is particularly However, when intact cells no substrate degradation, suggesting that the protease is not present on the exterior The ability of red cell membranes to degrade hemoglobin was subunits of the human red to the cell membrane 0 MEMBRANE VOLUME (ml) From www.bloodjournal.org by guest on July 31, 2017. For personal use only. BALLAS 878 4C , , ‘ AND BURKA I ID Li ID 4 a: H 30 Q (: Fig. of KSCN cxRBC membranes in degrading radioactive hemoglobin and hemoglobin chains at 37’C. Equal amounts of substrate were placed in the incubation system and assayed as described in the Methods section. The shaded area at the bottom of the figure shows the level of degradative activity in KSCN extracts of membrane-free hemolysate. tracts 3 of Activity . normal that the membrane-associated human beta chain. In order to ensure that Li - #{149} 2 / a: H I 0 .-..--.----. I .- .- - - a’ , / , Hb , ‘ ‘ -- 0 - - 4 8 2 TIME proteolytic the activity protease has activity 6 20 24 (hrs) a particular attributed affinity to the for the human red cell membrane was not due to contamination with other formed elements of the blood, an experiment was carried out in which the initial blood sample was enriched 40-fold with leukocytes. Before being separated into membrane and membranefree hemolysate human and blood from a was patient used enriched with in the with chronic difference between the rates contained was minor no added activity leukocytes, in degrading proteolytic leukocytes assay collected myelocytic system, leukemia. of substrate normal heparinized by differential degradation There centrifugation was no by the control significant samples, which and those that were enriched. In both samples hemoglobin and greater activity in breaking there down hemoglobin chains. Membrane-free hemolysate from either leukocyte-poor or leukocyte-enriched samples caused no substrate degradation. Thus, the proteolytic activity attributed to human erythrocyte membranes cannot be ascribed to contammating leukocytes. As shown membranes, 0.75-M 3, proteolytic from cell proteolytic activity in 0.75 extracts containing directed against of the membranes of red hemoglobin is confined approach susceptible [‘4C]leucine an energy-generating be extracted from by a final cell was used red cell of progressively when incubated findings confirm or its subunits, to the cell blood concentration membrane a period of 24 hr. Thiocyanate, did not cause degradation. These and alone that which membrane. to confirm that alpha and beta to the membrane protease. Intact red blood cells for 1 hr at 37#{176}C in order to label the hemoglobin. At the end of 1 hr the cells were washed membrane-free hemolysate and membranes were washed free of hemoglobin. Both suspension could hemolysates, M thiocyanate, different experimental chains are not equally were incubated with activity membrane-free Thiocyanate hemoglobin over of the substrates, is extractable A not thiocyanate. degraded with any red in Fig. but were system then in medium containing cold leucine; the were separated, and the membranes the membrane-free hemolysate and a reincubated in a Krebs-Ringer for 3 hr at 37#{176}C, and synthesized alpha and beta chains attached in the membrane chromatography of the globin chains on CM-52.’5 In confirmation the was fate buffer of the newly determined by of earlier studies From www.bloodjournal.org by guest on July 31, 2017. For personal use only. ERYTHROCYTE MEMBRANE PROTEASE ACTIVITY 879 10 V.) 2 NORMAL - 4 Fig. alpha 8 )( ‘S- ; . -.-.- . 6 c:* -‘--‘-.>.L------ ! __ to isolated , membranes for 3 hr at during incubation 37’C. Hemoglobin i: was labeled in the intact cell during an initial incubation for hr at 37’C. The membranes 3 1-#{149} C) - 2 -I 4 isolated and washed until of hemoglobin and then incubated in fresh buffer. At intervals during the second ,(‘_‘ 2 I,- I incubation C -‘ 0 ‘ I 60 120 T IM E there was virtually in the incubated alpha and the period beta of 240 medium, from the increase of labeled hemolysate. indicating that the membrane. Selective in the alpha/beta 0.60 to 1.22 during during chains remaining membrane hemoglobin The fate chains attached to the membrane incubation there was a slight tachment progressive pronounced human beta 80 the amounts of and beta chains attached to the were determined. alpha labeled . (m in ) no degradation membrane-free incubation from I loss to acid-soluble of the newly was not the same but insignificant reflected of beta attached the 3-hr period of incubation, longer incubations are more susceptible an fragments synthesized (Fig. 4). During increase in the degradation degradation ratio of chains red cell were free “t2 4 Amounts of labeled beta chains remain- 4. and ing attached #{149} and not disat- chains resulted in a to the membrane was more (Table 1). These findings suggest to degradation by the membrane-bound effect that that protease. DISCUSSION Although earlier mature erythrocytes human erythrocytes,’ proteolytic activity is confined solely studies erythrocyte enzymes.’5 activity in the soluble Table 1. had indicated that and suggested that the present studies in the mature erythrocyte to the membrane site, Alpha Recent portion / Beta 1 Ratio of Nascent Chains of Chase (hr) 3 After Pulse Labeling Alpha/Beta (cpm) 0 0.60 1 0.81 2 1.03 3 2 activity was present in studies by Hanash and Rucknagel found proteolytic of immature red blood cells, but pointed out that this Time Experiment proteolytic it was present in the stromal fraction of extend these findings and indicate that is absent from the cell cytoplasm and as is true of a large number of other 1.22 0 0.70 2 1.19 4 1.73 0 0.70 24 1.76 and Chase From www.bloodjournal.org by guest on July 31, 2017. For personal use only. 880 BALLAS activity did not was absent specifically immature protease red cells be from look the cytosol of mature erythrocytes.’6 for the presence of enzyme activity erythroid cells, no conclusions activity reported in this study of differing degrees of maturity necessary to determine Rucknagel is similar this. degree when directed However, enzyme since the Studies in rabbit reticulocytes indicate amino acid loses this ability to withstand meric studies present alpha hemoglobin reported hemoglobin that beta particular studies chains to here, molecules chains have susceptibility associates, membrane, of indicate and beta normal that also Hanash in isolated This the is circulating containing proteolytic a subunits of polypeptide more of is of low molecule. that hemoglobin intrinsic cellular are and the presence erythrocytes hemoglobin the chains, by hemoglobin erythrocyte.’7 substituted The stability these workers membranes of required of mature tetrameric with hemoglobin, known normal reported neither consistent activity.’8 the the here, system for activity. proteolytic activity against BURKA can be drawn as to whether or not the is also present in immature cells. Studies of separated by gradient centrifugation will to the one reported ATP or an energy-generating The membrane-associated Since in the AND susceptible than tetra- degradation in which by the degradation membrane-associated of the individual protease. The chains of nascent attached a particular of beta to isolated membranes was susceptibility to proteolytic chains explains the finding monitored, indicate degradation. This of DeSimone and who observed betas chains that after disappeared attachment of hemoglobin more rapidly than did S molecules alphaA chains.’9 to the Since the present studies, as well as those of DeSimone, were done in cells from patients with balanced globin synthesis, and there is no pool of free beta chains present in erythrocytes, this indicates molecule to the membrane that following the beta chain chain. This might occur either after breakdown of the molecule while into membrane. mode Since the membrane remains rate of degradation of attachment of relationships. The fact that exact attachment is degraded of the tetrameric more rapidly the molecule is still in the individual chains following of binding between individual to the hemoglobin hemoglobin the alpha tetrameric attachment form or to the hemoglobin unknown, it is not possible to determine of the beta chain is due to the particular hemoglobin than and whether spatial membrane or to specific subunits are degraded the the increased arrangements enzyme-substrate more rapidly than tetrameric hemoglobin suggests that the activity of the membrane-associated enzyme may have special application in conditions (such as the thalassemia syndromes) in which there is unbalanced globin synthesis. Two lines of evidence suggest that proteolytic enzymes play a role in protecting the erythrocyte against injury. First, with the cell of cells from the excess membrane patients alpha before with chains being 3-thalassemia produced lost from in 3-thalassemia the cell.2 Second, after labeling become during of the associated incubation hemoglobin with radioactive amino acids, the excess alpha chains are lost from the cell, presumably by proteolysis.7’t These findings suggest, but do not prove, that proteolytic enzymes within the red cell destroy excess hemoglobin chains and thus may protect the cells from the deleterious has been suggested effects that of attachment altered degrees contribute a degree of control to the thalassemia,79’2#{176} but there is no factual of these of proteolytic severity evidence chains to the cell activity of clinical to support with membrane.3 the red It cell may hemolytic syndromes this contention. in From www.bloodjournal.org by guest on July 31, 2017. For personal use only. ERYTHROCYTE The MEMBRANE factors responsible erythrocyte remain lular enzymes and may contribute removal PROTEASE for senescence poorly understood. alterations in both to this of senescent process.2’23 hemoglobin chains premature destruction to and death Decreases structure The erythrocytes system may be partially due membrane that is associated 881 ACTIVITY of membrane by the lesions macrophages maintenance The remains activity poorly the membrane of red blood in cells3 thalassemia supports globin significance synthesis understood. the soluble fraction of human erythroid cell istics cytosol of rabbit from that found in the than one proteolytic Further experimental enzymes are erythroid enzyme studies responsible cell and unbalanced globin synthesis on localization of a protease to the that the red cell membrane of hemoglobin and proteolytic and thalassemic activity also erythroid contributes enzymes activity erythroid the life differs in several reticulocytes,’t there membrane significantly and of of the the be more erythroid cell. which enzyme or its subunits red mature participates charactermay within in ameliorating span cell.9 in human erythroid cells that has been reported in precursors9 play to Since in the maturing enzymes to the cell of excess contributes possibility. of hemoglobin role these to eventual of protein attachment within the maturing mammalian will be necessary to define precisely for degradation what lead reticuloendothelial syndromes this of the proteolytic Since the proteolytic circulating of intracelcell membrane that to attachment of small amounts with cell aging.’ The fact that of balanced and normal of the activity has been found in crude lysates of both normal cell precursors, it has been suggested that this proteolytic to the the in the concentrations and function of the the cell. the effects of However, erythrocyte does in the intracellular the indicate catabolism its subunits. REFERENCES 1. Sears of DA, Friedman intracellular membrane Heinz i, protein during bodies. White to incubation: i Lab 2. Bargeilesi Clin A, DR: the The semia production Med 86:722, Pontremoli C, ity 1975 Menini F: Excess zygous f3-thalassemia and its removal from cell cytoplasm. Eur i Biochem 3:364, 1968 3. Nathan 1)6, Gunn consequences sis. Am 41:815, 4. Morrison enzymes of the i Biol 5. elements GL, J, Robinson 6. lases. cyte 7. Proteolytic H: of human blood. 1953 Kocholaty SL: WF, Cooper A proteinase from Biochim Biophys erythrocyte membranes. 212:126, synthe- DA, human Acta II. Ri, Bosmann Proteinase HB: activities plasma membranes. Bank A, O’Donnell i Membr JV: Red cell hydro- in human Biol erythro7:1, 1972 Intracellular loss of free chains in 9-thaiassemia. Nature 222:295, in USA Wood WG, Stamotoyannopoulos G: Globin Invest 5, Rucknagel red cell 1975 Proteolytic activ- DL: precursors. of fl-thalas55:567, Proc NatI Acad Sci Di: The (in press) 10. Dodge preparation Biochem iT, Micheli and chemical globin-free ghosts Biophys 11. Lowry of C, human 100:119, OH, Hanahan characteristics 12. erythrocytes. Rosebrough reagent. i Biol Bulova SI, of hemo- Ni, Chem Lau 193:265, Burka 13. Winterhaiter KH, properties and ER: 1951 Biosynthesis 239:3699, lysate Biophys Abnormal of ribosomes 1970 ER: Prepara- recombination subunits. i of Biol Chem 1964 14. Adamson Factors Huehns specific AL, with the Folin nonglobin protein by membrane-bound in reticulocytes. i Biol Chem 245:4907, tion, Arch 1963 Ri: Protein measurement phenol 15. 1969 8. normoblasts i Clin alpha-beta-globin 1970 Bernachi Hanash Randall Neurath Moore Gray hemoglobin WG, 200:39, the red 1966 formed Chem in homo- RBL: Thalassemia: The of unbalanced i Med synthesis C, in fractionated heterozygotes. 9. of Conconi blood of a-globin synthesis Binding erythrocyte 5, affecting systems from 125:671, Clegg Herbert the iB, human rate E, Godchaux of protein reticulocytes. W: synthesis Arch in Biochem 1968 Naughton hemoglobins. MA, Weatherall Separation D: and From www.bloodjournal.org by guest on July 31, 2017. For personal use only. BALLAS 882 characterization of the chromatography, and variants, Mol Hb Biol 16. Blood 17. Chesapeake 19:91, Schrier 50:227, and chains of Hb two by i (Bangkok). i sickle erythrocyte and membrane clinical AL, St iohn AC: in mammalian iD, Goldberg AL: Proc Natl 19. of abnormal Acad DeSimone proteins Sci USA i, 74:54, Adams enzyme ATP- 1977 Schaeffer newly in sickle A: The synthesized cell anemia 35:373, thalassemia and 1977 syndromes. G: life 16:257, Bishop Blood of modifications the erythrocyte. Gastel C: 23. i: Danon Van during 1), Perk tion of erythrocytes tron microscopic 59:117, 1962 Ital i 1967 C, activity Biochemical span reticuiocyte and red cell aging. Haematoiogica in reticulocytes. iG, the 22. 1976 dependent proteolytic system responsible for the degradation loss of Br i Haematoi Fornaini Biochem bacterial A soluble trait. Bank 21. Intracellular 45:747, rapid S molecules BURKA 5 1:369, 1978 correlates. and Biochem Rev for cell during degradation Etlinger Evidence haemoglobin new 20. Human status cells: Part 2. Annu 18. beta 1977 Goldberg protein and 1966 SL: Current enzymes: alpha determination AND 3:29, 1969 population in domestic animals. i Cell in maturation K: Age study. Changes Comp distribuAn elec- Physiol From www.bloodjournal.org by guest on July 31, 2017. For personal use only. 1979 53: 875-882 Protease activity in the human erythrocyte: localization to the cell membrane SK Ballas and ER Burka Updated information and services can be found at: http://www.bloodjournal.org/content/53/5/875.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.