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1.
Clin Exp Immunol ; 106(2): 323-8, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8918580

ABSTRACT

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). This was initially identified as a 64-65 kD molecule according to migration in gels after immunoprecipitation from pancreatic islets. We studied the antigenicity of two different radiolabelled preparations of GAD, derived either by affinity purification from porcine brain and known to contain GAD 65 and GAD 67, or by expression from a cDNA for human GAD 65 by rabbit reticulocyte lysate (RRL). Radiolabelled immunoprecipitated pellets from the reaction of potent antisera to GAD from patients with IDDM were examined by autoradiography after SDS-PAGE under reducing or non-reducing conditions. Also, preparations of porcine brain GAD were "depleted' of GAD by exposure to antisera, and then similarly re-examined. Autoradiography of radiolabelled GAD either affinity purified from porcine brain, or expressed by RRL, showed that the immunoprecipitated protein migrated under non-reducing conditions according to a M(r) of approximately 110-130 kD, corresponding to dimeric forms of monomeric GAD of approximately 55-65 kD. Depletion by immunoprecipitation of this minor higher M(r) component from preparations of GAD left, in the supernatant, an abundance of GAD of M(r) 64-65 kD corresponding to monomer that was completely non-reactive with potent IDDM sera. We conclude that IDDM sera react with the GAD molecule in a dimeric (or oligomeric) form. Our findings have general connotations for self-tolerance and autoimmunity.


Subject(s)
Diabetes Mellitus, Type 1/immunology , Epitopes/immunology , Glutamate Decarboxylase/immunology , Isoenzymes/immunology , Animals , Autoantibodies/immunology , Autoradiography , Brain/enzymology , Cross Reactions/immunology , Dimerization , Electrophoresis, Polyacrylamide Gel , Humans , Precipitin Tests , Rabbits , Recombinant Proteins , Swine
2.
Diabet Med ; 11(9): 866-71, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7705024

ABSTRACT

The frequency of antibodies to glutamic acid decarboxylase (GAD) and insulin (IAA) in presymptomatic Type 1 diabetes mellitus with a positive test for antibodies to islet cell antigen (ICA) was examined. Thirty-two persons positive for ICA (> 10 JDF units) were tested 2 to 48 months before their ascertained onset of Type 1 diabetes. ICA was quantitated by immunofluorescence as JDF units, anti-GAD by radioimmunoprecipitation and anti-insulin by radioimmunoassay. There was a positive test for anti-GAD in 25 (78%), and for IAA in 23 (72%), of the 32 prediabetic ICA-positive subjects. Stratification according to age at the onset of diabetes showed differing frequencies of anti-GAD and IAA in the prediabetic stage. Thus the positivity rate for anti-GAD for 18 subjects older than 10 years at onset of diabetes was 83%, and for 14 aged 10 or younger at onset was 71%; conversely, the rate for IAA for 18 subjects older than 10 at onset was 56% and for 14 aged 10 or less at time of onset was 93% (p = 0.01). The frequency of anti-GAD was higher in females (88%) than males (71%) whereas the frequency of IAA was higher in males (82%) than in females (60%). Since autoantibodies to GAD and insulin occur in presymptomatic Type 1 diabetes with differences in frequencies by age and gender, the stimuli to autoimmunity may operate differently at different ages, and may also be gender-related.


Subject(s)
Autoantibodies/blood , Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/immunology , Insulin Antibodies/blood , Islets of Langerhans/immunology , Adolescent , Adult , Age Distribution , Age of Onset , Child , Child, Preschool , Diabetes Mellitus, Type 1/diagnosis , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Sex Distribution
3.
Am J Epidemiol ; 140(8): 683-90, 1994 Oct 15.
Article in English | MEDLINE | ID: mdl-7942770

ABSTRACT

Diabetes mellitus is a heterogeneous disease. The better classification of types of diabetes mellitus among adults will improve epidemiologic studies of determinants of risk factors and genetic host susceptibility. Recently, an antibody to a specific enzyme, glutamic acid decarboxylase, has been closely linked to insulin-dependent diabetes mellitus. Sera were collected at baseline between 1972 and 1974 from initially nondiabetic participants in the Multiple Risk Factor Intervention Trial. After approximately 18 years of frozen storage, the serum samples were tested for antibodies to glutamic acid decarboxylase (anti-GAD) in 175 men who developed diabetes and 352 matched controls who did not develop diabetes during the 6-year follow-up. Nine of the 527 samples tested had elevated (19 or more units) titers of anti-GAD. Six of the nine men with elevated anti-GAD subsequently developed diabetes, and three of these six were ultimately placed on insulin therapy. These data suggest that elevated levels of anti-GAD may be a prospective marker for the subsequent development of insulin-dependent diabetes mellitus. The measurement of anti-GAD is relatively easy, can be performed in stored serum specimens, and may be used in epidemiologic studies to enhance the understanding of the determinants of diabetes mellitus.


Subject(s)
Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/immunology , Adult , Antibodies/blood , Biomarkers/blood , Case-Control Studies , Clinical Trials as Topic , Coronary Disease/prevention & control , Diabetes Mellitus/epidemiology , Diabetes Mellitus/immunology , Diabetes Mellitus, Type 1/enzymology , Diabetes Mellitus, Type 1/epidemiology , Humans , Male , Middle Aged , Risk Factors , Seroepidemiologic Studies
4.
Clin Immunol Immunopathol ; 71(1): 53-9, 1994 Apr.
Article in English | MEDLINE | ID: mdl-7511084

ABSTRACT

The frequency of antibodies to GAD (anti-GAD) in insulin-dependent diabetes mellitus (IDDM) varies greatly according to the type of assay employed. We therefore examined the immunoassay characteristics of diabetic sera using GAD purified from porcine brain and shown to contain both isoforms. Sera from 38 patients with IDDM, including 1 patient with both stiff-man syndrome (SMS) and IDDM, were studied for anti-GAD by radioimmunoprecipitation (RIP), Western blotting, and dot-blotting. The sera were selected according to reactivity in the RIP assay. There was a good correlation between potency of the RIP reaction at the screening dilution of 1:2 and the endpoint dilution in the assay which ranged from 1:2 to 1:30,000 for IDDM sera, and 1:300,000 for the SMS serum. Of the 38 sera positive for anti-GAD by RIP, only 6 had antibodies detectable by Western blotting, and all gave an RIP titer of at least 1:250. The low frequency of antibodies by Western blotting was explicable by denaturation of the antigen. Thus, using a dot-blotting assay in which reactivity to untreated "native" GAD was compared with reactivity to GAD after denaturation by reduction with 2-mercaptoethanol and boiling, 20 of the 38 IDDM sera reacted unequivocally with the native GAD compared with only 2 that reacted with denatured GAD after reduction and boiling. The sera were tested for their capacity to inhibit the catalytic activity of GAD, but only the high-titer serum from the patient with SMS did so. Our study further validates the RIP assay for anti-GAD and establishes that anti-GAD exists in IDDM over a wide range of titers that correlate with other assay characteristics, and also indicates that the conformational autoantibody epitope on GAD is susceptible to alteration under denaturing conditions.


Subject(s)
Autoantigens/immunology , Glutamate Decarboxylase/immunology , Adolescent , Adult , Animals , Autoantibodies/blood , Blotting, Western , Child , Child, Preschool , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/enzymology , Enzyme-Linked Immunosorbent Assay , Epitopes , Glutamate Decarboxylase/antagonists & inhibitors , Glutamate Decarboxylase/blood , Humans , Immunoblotting , Infant , Middle Aged , Precipitin Tests , Protein Denaturation , Radioimmunoassay , Stiff-Person Syndrome/blood , Stiff-Person Syndrome/enzymology , Swine
5.
Pediatr Res ; 34(6): 785-90, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8108194

ABSTRACT

Antibodies to glutamic acid decarboxylase (GAD), previously known as the 64-kD pancreatic islet cell autoantigen, are an important serologic marker of insulin-dependent diabetes mellitus (IDDM). Antibodies to GAD (anti-GAD) were examined in sera from Australian children with newly diagnosed IDDM (within 1 mo of diagnosis), IDDM of longer duration (mean +/- SD, 4.8 +/- 3.3 y), and in first-degree relatives, using a radioimmuno-precipitation assay with purified porcine brain GAD as antigen. Antibodies to islet cell cytoplasmic antigens (ICAb) were tested concurrently. The frequency of anti-GAD was not significantly different in children with newly diagnosed IDDM (31 of 42, 74%) and with IDDM of longer duration (14 of 21, 67%), whereas ICAb were present more frequently in children with newly diagnosed IDDM (64%) than in those with longer duration IDDM (14%). In all, 90% of children with newly diagnosed IDDM had either anti-GAD or ICAb, whereas only 48% had both. For the 77 first-degree relatives, the frequency of anti-GAD was 2% (one of 44) in parents and 6% (two of 33) in siblings; ICAb were not detected in any of these relatives. The presence of anti-GAD in the majority of children with IDDM, irrespective of the duration of their disease, represents a useful diagnostic marker for IDDM, and should be of value in ascertaining individuals at risk for developing IDDM.


Subject(s)
Autoantibodies/blood , Diabetes Mellitus, Type 1/enzymology , Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/immunology , Adolescent , Adult , Alleles , Australia , Autoantigens , Child , Child, Preschool , Diabetes Mellitus, Type 1/genetics , Female , HLA-DQ Antigens/genetics , HLA-DQ beta-Chains , Humans , Islets of Langerhans/enzymology , Islets of Langerhans/immunology , Male , Middle Aged , Radioimmunoprecipitation Assay
6.
J Diabetes Complications ; 7(1): 1-7, 1993.
Article in English | MEDLINE | ID: mdl-8481544

ABSTRACT

Our objective was to ascertain the frequency of antibodies to glutamic acid decarboxylase (GAD) in Europids and four Asian ethnic groups with insulin-dependent diabetes mellitus (IDDM) to gain insight into why the prevalence and incidence of IDDM varies so widely among ethnic and/or geographically diverse population groups. The subjects in this study were Europid (n = 49), Japanese (n = 16), Thai (n = 7), Korean (n = 21), and Chinese (n = 13) persons with IDDM with a duration ranging from 5 to 14 years. There were similar numbers of healthy controls matched for each ethnic group. A validated radioimmunoprecipitation assay used GAD from pig brain radiolabeled with 125I using chloramine T. Islet cell cytoplasmic antibodies measured by indirect immunofluorescence were expressed as Juvenile Diabetes Foundation units. The prevalence of antibodies to GAD, compared with Europids (63%), was much lower in all Asian populations with IDDM: Japanese (31%), Thai (29%), Korean (5%), and Chinese (27%). The mean level of antibodies to GAD, however, among diabetics from each population who gave a positive reaction, was similar. For all groups, the prevalence of antibodies to GAD was much higher than that of islet cell cytoplasmic antibodies. Almost all IDDM subjects positive for islet cell antibodies had antibodies to GAD, but the converse did not hold. A radioimmunoprecipitation assay for antibodies to GAD applied to serum from subjects with IDDM in various ethnic groups showed that Europids with IDDM had a much higher prevalence of such antibodies than did Asians. This held for all ethnic groups, and particularly Koreans. Thus, among different populations, there may be etiologic heterogeneity of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Asian People , Autoantibodies/blood , Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/immunology , White People , Diabetes Mellitus, Type 1/enzymology , Diabetes Mellitus, Type 1/ethnology , Humans , Incidence , Prevalence
7.
Diabetes ; 41(4): 548-51, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1607079

ABSTRACT

Insulin-dependent diabetes mellitus (IDDM) is marked by circulating antibodies to a 64,000-M(r) islet cell antigen identified as glutamic acid decarboxylase (GAD). We describe a radioimmunoprecipitation assay with GAD isolated from pig brain. The sera tested were from 80 patients with IDDM including 26 with disease of recent onset and 54 with disease of longer duration (3-42 yr), 20 with non-insulin-dependent diabetes mellitus (NIDDM), and 55 nondiabetic subjects. Conventional assays for islet cell cytoplasmic antibodies were performed concurrently. The level of antibody in serum was expressed in units based on percentage reactivity of a standard reference serum. The frequency of antibody to GAD in IDDM was 69% in short-duration cases and 59% in long-duration cases. The latter was substantially higher than the frequency of islet cell cytoplasmic antibody. Antibodies to GAD were elevated (means +/- 3 SD) in 5% NIDDM cases and in none of the nondiabetic subjects. A simple laboratory test with a defined autoantigen has substantial implications for population screening and early diagnosis of IDDM and for better understanding of its pathogenesis.


Subject(s)
Antibodies/analysis , Diabetes Mellitus, Type 1/diagnosis , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 2/diagnosis , Diabetes Mellitus, Type 2/immunology , Glutamate Decarboxylase/immunology , Adolescent , Adult , Aged , Antibodies/immunology , Autoantibodies/analysis , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 2/pathology , Diagnosis, Differential , Female , Humans , Islets of Langerhans/chemistry , Islets of Langerhans/immunology , Islets of Langerhans/pathology , Male , Middle Aged , Radioimmunoprecipitation Assay
8.
J Cell Biol ; 110(3): 789-801, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2407741

ABSTRACT

Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.


Subject(s)
Cell Adhesion , Endothelium, Vascular/physiology , Extracellular Matrix/physiology , Integrins/physiology , Oligopeptides/pharmacology , Acid Phosphatase/metabolism , Amino Acid Sequence , Animals , Aorta , Cattle , Cell Adhesion/drug effects , Cell Movement , Cells, Cultured , Fibrinogen/physiology , Fibronectins/physiology , Fluorescent Antibody Technique , Laminin/physiology , Molecular Sequence Data , Oligopeptides/chemical synthesis , Oligopeptides/metabolism
9.
J Biol Chem ; 264(7): 4033-7, 1989 Mar 05.
Article in English | MEDLINE | ID: mdl-2783929

ABSTRACT

The composition of the intramembranous domains of many receptors are remarkably uniform, yet there is evidence that many transmembrane proteins associate together to form specific noncovalent homo- or heterocomplexes within the membrane. We have synthesized peptides corresponding to transmembrane domains of glycophorin A, glycophorin C, and the interleukin 2-receptor Tac antigen to study the interactions between transmembrane domains in vitro. Synthetic transmembrane glycophorin A peptide formed a complex with native glycophorin and glycoproteins of erythrocyte and K562 cell membranes that was reversible, specific, and could be demonstrated in a natural bilayer system in the absence of detergents. Synthetic glycophorin C and interleukin 2-receptor Tac antigen transmembrane peptides, although similar in amino acid composition, did not interact with glycophorin and did not inhibit the binding of the synthetic glycophorin A transmembrane peptide to native glycophorin. It is proposed that the transmembrane segments of receptor proteins contain not only the structural information necessary for insertion and anchoring but specific binding sites that mediate interactions between transmembrane glycoproteins.


Subject(s)
Glycophorins , Membrane Glycoproteins , Receptors, Interleukin-2 , Sialoglycoproteins , Amino Acid Sequence , Erythrocyte Membrane , Humans , In Vitro Techniques , Liposomes , Macromolecular Substances , Molecular Sequence Data , Peptide Fragments , Protein Binding
10.
Blood ; 67(1): 141-51, 1986 Jan.
Article in English | MEDLINE | ID: mdl-3940543

ABSTRACT

An abnormal alpha subunit of erythrocyte spectrin has been described in hereditary pyropoikilocytosis (HPP), a rare hemolytic anemia characterized by erythrocyte budding and fragmentation. In HPP spectrin, the N terminal domain of the alpha subunit (alpha I T80) shows increased susceptibility to tryptic digestion, resulting in cleavage to a 50,000-d peptide, presumably due to a change in primary structure of the alpha I domain which alters conformation and generates the new cleavage site. The functional result of this conformational alteration is marked impairment of spectrin oligomer formation in vitro, consistent with the established role of alpha I T80 in spectrin self-association. In the present study, we demonstrate an abnormal spectrin alpha subunit in two kindreds with hereditary elliptocytosis (HE) that is qualitatively identical to HPP spectrin. Clinical expression of HE in these families ranges from mild elliptocytosis without hemolysis to severe poikilocytic hemolytic anemia clinically resembling HPP. In all affected individuals, a fraction of alpha I T80 is abnormal, as shown by its cleavage during mild tryptic digestion to the 50 kd peptide described in HPP; the fraction of alpha I T80 affected is directly proportional to the severity of clinical expression of HE. Spectrin oligomer formation is likewise impaired to a degree which correlates with hematologic disease. One of the HE kindreds studied demonstrated polymorphism in the spectrin alpha II domain, previously described as a frequent occurrence in blacks. This family also demonstrates a variant alpha III domain in spectrin that has not previously been described. We conclude that the abnormality in the alpha I domain originally described in HPP spectrin is shared by a subset of patients with HE; the severity of clinical expression, ranging from mild nonhemolytic HE to poikilocytic hemolytic anemia, is related to the fractional quantity of the alpha subunit that is affected.


Subject(s)
Elliptocytosis, Hereditary/blood , Spectrin/analysis , Adolescent , Adult , Anemia, Hemolytic, Congenital/blood , Female , Humans , Male , Middle Aged , Polymers/metabolism , Trypsin/pharmacology
11.
J Clin Invest ; 73(4): 973-9, 1984 Apr.
Article in English | MEDLINE | ID: mdl-6707213

ABSTRACT

Restricted tryptic digestion of erythrocyte spectrin at 4 degrees C followed by two-dimensional (isoelectric-focusing/sodium dodecyl sulfate) polyacrylamide electrophoresis yields highly reproducible maps of approximately 50 peptides with molecular weights between 80,000 and 12,000. Based on molecular weight and isoelectric point (pI), each unique alpha- and beta-subunit domain can be identified and compared with spectrin peptides from other individuals. The alpha-subunit of spectrin from 60 Caucasian donors contains a 46,000-mol-wt tryptic domain, called alpha II-T46, Type 1; more extensive tryptic digestion of this domain generates peptides with molecular weights of 35,000, 30,000, 25,000, and 16,000. Spectrin from 29 of 37 black donors representing 14 kindreds shows variation in the molecular weight and/or pI of peptides from the alpha II domain. In the most common form, Type 2, alpha II tryptic peptides are increased in molecular weight by 4,000, and the pI becomes more basic. Other alpha II variants are characterized by either the 4,000 increase in molecular weight (Type 3) or by the basic shift in pI (Type 4). When limit peptide maps of intermediate-sized tryptic and CNBr peptides from the alpha II-domain Types 1 and 2 are compared, a consistent alteration in the chromatographic mobility of one limit peptide is observed. Polymorphism in the alpha II subunit of spectrin did not itself produce anemia, nor did it appear to alter the expression of an underlying hereditary spherocytosis or elliptocytosis. In six family studies, the alpha II 46,000-mol-wt variations observed were consistent with Mendelian inheritance.


Subject(s)
Genes , Polymorphism, Genetic , Spectrin/genetics , Electrophoresis, Polyacrylamide Gel , Elliptocytosis, Hereditary/blood , Elliptocytosis, Hereditary/genetics , Erythrocytes, Abnormal/analysis , Genetic Variation , Humans , Molecular Weight , Peptides/blood , Spherocytosis, Hereditary/blood , Spherocytosis, Hereditary/genetics
12.
J Clin Invest ; 71(6): 1867-77, 1983 Jun.
Article in English | MEDLINE | ID: mdl-6863544

ABSTRACT

The structural and functional properties of spectrin from normal and hereditary pyropoikilocytosis (HPP) donors from the two unrelated families were studied. The structural domains of the spectrin molecule were generated by mild tryptic digestion and analyzed by two-dimensional electrophoresis (isoelectric focusing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The alpha I-T80 peptide (Mr 80,000) is not detectable in two related HPP donors; instead, two new peptides (Mr 50,000 and 21,000) are generated and have been identified as fragments of the normal alpha I-T80. A third sibling has reduced levels of both the normal alpha I-T80 and the two new peptides. A similar analysis of spectrin from another HPP family indicates that their spectrins contain reduced amounts of the alpha I-T80 and the 50,000 and 21,000 fragments of the alpha I domain. The HPP donor also has other structural variations in the alpha I, alpha II, and alpha III domains. The alpha I-T80 domain of normal spectrin has been shown to be an important site for spectrin oligomerization (J. Morrow and V.T. Marchesi. 1981. J. Cell Biol. 88: 463-468), and in vitro assays indicate that HPP spectrin has an impaired ability to oligomerize. Ghost membranes from HPP donors are also more fragile than membranes from normal erythrocytes when measured by ektacytometry. In both the oligomerization and fragility assays, the degree of impairment is correlated with the amount of normal alpha I-T80 present in the spectrin molecule. We believe that a structural alteration in the alpha I-T80 domain perturbs normal in vivo oligomerization of spectrin, producing a marked decrease in erythrocyte stability.


Subject(s)
Anemia, Hemolytic, Congenital/genetics , Erythrocytes/physiology , Membrane Proteins/physiology , Spectrin/physiology , Adult , Anemia, Hemolytic, Congenital/blood , Erythrocytes/analysis , Female , Hot Temperature , Humans , Macromolecular Substances , Male , Peptide Fragments/analysis , Protein Denaturation , Spectrin/analysis , Trypsin
14.
J Biol Chem ; 257(15): 9093-101, 1982 Aug 10.
Article in English | MEDLINE | ID: mdl-7096353

ABSTRACT

Proteolytic susceptibility has been used to probe the structure of human erythrocyte spectrin. Nine unique polypeptide segments have been defined by mild trypsin digestion (0 degrees C) and analyzed by two-dimensional peptide mapping techniques. These peptide segments, referred to operationally as chemical domains, exhibited varying degrees of sensitivity to further proteolytic cleavage. One region (beta I) which contained the phosphorylated amino acids of the beta subunit was quite sensitive to proteolysis and was rapidly degraded to numerous small peptides. Overlap peptides produced by enzymatic and chemical cleavages were used to align each domain in the appropriate spectrin subunit. The molecular weights of the largest unique peptides from both subunits sum to the approximate weight of the intact molecule. Similarly, summation of the two-dimensional peptide maps of the intermediate sized peptides approximates the two-dimensional maps of the intact spectrin subunits, indicating that most or all of the molecule is represented. These results suggest that spectrin is composed of multiple, ordered, largely alpha-helical domains that are connected by small protease-sensitive segments. A comprehensive structural model is presented.


Subject(s)
Erythrocytes/analysis , Membrane Proteins , Models, Chemical , Spectrin , Electrophoresis, Cellulose Acetate , Electrophoresis, Polyacrylamide Gel , Humans , Macromolecular Substances , Membrane Proteins/analysis , Molecular Weight , Peptide Fragments/analysis , Spectrin/analysis , Trypsin/metabolism
15.
J Biol Chem ; 257(15): 9102-7, 1982 Aug 10.
Article in English | MEDLINE | ID: mdl-7096354

ABSTRACT

A library of nine monoclonal antibodies which bind to the alpha subunit of human erythrocyte spectrin has been established. The specificity of these antibodies confirms the alignment and uniqueness of each of the five previously identified peptide domains in this subunit and establishes the identity of additional smaller proteolytic peptide fragments. This immunochemical approach is complementary to the identification of peptide relationships by two-dimensional chymotryptic peptide mapping, and the results of both methods are in complete agreement.


Subject(s)
Antibodies, Monoclonal/immunology , Membrane Proteins/analysis , Spectrin/analysis , Humans , Macromolecular Substances , Molecular Weight , Peptide Fragments/analysis , Peptide Fragments/immunology , Spectrin/immunology , Trypsin/metabolism
16.
Proc Natl Acad Sci U S A ; 77(11): 6592-6, 1980 Nov.
Article in English | MEDLINE | ID: mdl-6935670

ABSTRACT

Isolated human erythrocyte spectrin is a dimer of two unique polypeptide chains. The dimer (alpha beta) undergoes reversible salt- and temperature-dependent association to form (alpha beta)2 tetramers. Spectrin also binds with high affinity to a protein receptor on the cytoplasmic surface of erythrocyte membrane vesicles. By cleavage of spectrin at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid, a 50,000-dalton peptide fragment has been isolated which inhibits the binding of spectrin to erythrocyte membrane vesicles. This peptide arises from a terminal region of the beta chain. An 80,000-dalton peptide generated by restricted trypsin digestion binds preferentially to dimeric spectrin. This peptide arises from a terminal portion of the alpha chain. Multiple peptides involved in noncovalent associations between the chains have also been identified. These associations indicate that the two subunits of spectrin are aligned parallel to one another and that the tetramer formation site and the high-affinity membrane binding site are in close proximity to one another.


Subject(s)
Erythrocytes/ultrastructure , Membrane Proteins , Spectrin , Binding Sites , Erythrocyte Membrane/metabolism , Humans , Macromolecular Substances , Membrane Proteins/metabolism , Peptide Fragments/metabolism , Spectrin/metabolism
17.
Proc Natl Acad Sci U S A ; 77(10): 5673-7, 1980 Oct.
Article in English | MEDLINE | ID: mdl-7003593

ABSTRACT

Digestion of purified human erthrocyte spectrin with proteolytic enzymes at 0 degrees C results in the production of intermediate-size peptides that resist further cleavage at 0 degrees C. By two-dimensional peptide analysis of these intermediate peptides it has been determined that five unique peptides are produced by tryptic cleavage of the alpha subunit of spectrin (band 1); these have apparent molecular weights of 80,000, 46,000, 46,000, 41,000, and 30,000 and account for 97% of the alpha subunit. Similarly, four unique peptides having apparent molecular weights of 74,000, 65,000, 33,000, and 38,000 account for 90% of the beta subunit (band 2). By examining larger peptide fragments, the linear alignment of the unique peptides along each of the spectrin subunits has been established. These results indicate that spectrin is composed of two nonidentical subunits, each containing multiple proteolytically resistant domains. These domains, which may be largely alpha-helical, seem to be connected by small protease-sensitive segments. The proteolytic resistance of these domains is not influenced by the multimeric state of the spectrin molecule.


Subject(s)
Erythrocyte Membrane/ultrastructure , Erythrocytes/ultrastructure , Membrane Proteins/metabolism , Peptide Hydrolases/metabolism , Spectrin/metabolism , Chymotrypsin/metabolism , Erythrocytes/metabolism , Humans , Peptide Fragments/analysis , Trypsin/metabolism
19.
J Parasitol ; 63(3): 476-85, 1977 Jun.
Article in English | MEDLINE | ID: mdl-864566

ABSTRACT

A method for isolating an enriched preparation of tegumental brush border from the tapeworm, Hymenolepis diminuta, is described. Combining incubation of whole tapeworms in Krebs-Ringer/tris-maleate solution containing a hemolytic saponin, low shear-force agitation, and differential centrifugation, a pellet is obtained at 2,500 g which contains a significant concentration of surface brush border. The content of brush border in this fraction is identified by the presence of numerous microvilli, increased specific radioactivity after surface tagging with 3H-Concanavalin A, and relatively little mitochondrial contamination (succinic dehydrogenase). Based on morphological criteria, fractions sedimenting with greater force contain dense vesicles and mitochondria from the outer portion of the tegument.


Subject(s)
Cestoda/cytology , Hymenolepis/cytology , Mitochondria , Saponins
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