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PubMed1987

Identification of spectrin-related peptides associated with the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 kinase and of Mr 95,000 peptide that appears to be the catalytic subunit.

An isolation procedure for the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 (eIF-2 alpha) kinase is described which yields different fractions with kinase activity. Each is associated with a different spectrin-related peptide as identified by anti-spectrin monoclonal antibodies. The most abundant of these peptides is the Mr 90,000 species characterized previously (Kudlicki, W., Fullilove, S., Kramer, G., and Hardesty, B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5332-5336). Association with the spectrin-related peptides appears to account for the heterogeneity of the enzyme during its isolation and for its highly asymmetric structure. Isolated alpha or beta spectrin subunits as well as the separated homogeneous Mr 90,000 peptide cause an increase in the initial rate of eIF-2 alpha phosphorylation that is related to a decrease in Km with little or no effect on Vmax for the phosphorylation reaction. Fractionation of highly purified eIF-2 alpha kinase preparations using affinity chromatography on monoclonal anti-spectrin antibodies has separated eIF-2 alpha kinase activity from the Mr 100,000 phosphopeptide which copurifies with the kinase during all other purification steps. A Mr 95,000 peptide, detectable only by photoaffinity labeling with 8-azido-[alpha 32P]ATP, is shown to be distinct from the Mr 100,000 phosphopeptide and appears to be the catalytic subunit of the eIF-2 alpha kinase.

Animals↗

PubMed1987

A new variant of the alpha subunit of spectrin in hereditary elliptocytosis.

A kindred is described in which two brothers with a poikilocytic variant of hereditary elliptocytosis (HE) were found to have a defect of spectrin dimer association and a decreased spectrin-band 3 ratio. Two-dimensional gel electrophoresis of limited tryptic digests of their spectrin revealed decreased amounts of the alpha I domain when compared with control digests and the appearance of two major peptides with mol wts of 43,000 and 42,000 and isoelectric points (5.75 to 5.85) more basic than the alpha I domain. Tryptic digests of spectrin from the asymptomatic mother of the two brothers were normal. Immunoblots of the two-dimensional gels using an antiserum to the alpha I domain revealed that the 43,000- and 42,000-dalton peptides were derived from the alpha I domain, along with a series of lower mol wt peptides, some of which were below the detection limits of Coomassie blue-stained gels. Limit chymotryptic maps of 125I-labeled tryptic peptides confirmed that the 43,000- and 42,000-dalton peptides were derived from the alpha I domain. This kindred represents a new structural variant of spectrin in HE in that the major abnormal tryptic peptides derived from the alpha I domain have lower mol wts and more basic isoelectric points than hitherto described.

Adult↗

PubMed1985

A new abnormal variant of spectrin in black patients with hereditary elliptocytosis.

Seven black patients with mild hereditary elliptocytosis (HE) from five unrelated families were studied. The erythrocytes of these patients exhibited an abnormal thermal sensitivity (between 45 degrees C and 47 degrees C instead of 49 degrees C). An important defect of spectrin dimer self-association was detected in two ways: (1) the proportions of spectrin dimer (SpD) extracted from membranes at 4 degrees C under low ionic strength conditions were increased between 25% and 56% (normal value 15% +/- 2%); (2) the spectrin dimer----tetramer conversion in solution were defective with an association constant value between 0.4 and 2.4 X 10(5) M-1 for a normal value of 6 +/- 0.4 X 10(5) M-1. Spectrin (Sp) from HE patients and normal volunteers (32 black and 22 white subjects) was submitted to limited tryptic digestion, followed by one- or two-dimensional separation of the peptides. Peptide patterns of crude Sp from all seven HE patients exhibited a marked and reproducible decrease in 80,000-dalton peptide (previously identified as the dimer-dimer interaction domain of the alpha-chain) and a concomitant appearance of a novel 65,000-dalton peptide. A minor fragment at 28,000 daltons was also decreased. Tryptic digestion of HE spectrin dimer and tetramer (SpT), isolated after the SpD self-association procedure in solution, revealed modifications (decrease in the 80,000-dalton peptide and presence of a 65,000-dalton peptide) predominantly in HE SpD when peptide patterns of HE SpT were quite similar to control SpT patterns. Immunoblots with anti-alpha-chain antibodies revealed that the 65,000-dalton peptide derived from the alpha-chain. Kinetic studies of Sp digestion showed that the 65,000-dalton peptide did not result from further digestion of a 74,000 intermediate and was not a precursor of 46,000- to 50,000-dalton peptides. These results show a new structural defect of Sp-alpha-chain, associated with a defective Sp dimer self-association in HE.

Adult↗

PubMed1985

Rapid mobility of motile varicosities and inclusions containing alpha-spectrin, actin, and calmodulin in regenerating axons in vitro.

Time-lapse video recording was used to investigate the bidirectional movements of motile varicosities and intervening phase-dense (IPD) inclusions associated with axons of goldfish retinal ganglion cells regenerating in vitro. In addition, analyses of fine structure and immunocytochemical distributions of alpha-spectrin (fodrin), actin, and calmodulin in axonal fields were undertaken. Varicosities and IPD inclusions undergo saltations in a random manner at mean rates of 0.218 and 3.33 micron/sec, respectively. Experiments involving calcium antagonists or depletion of internal calcium stores resulted in an arrest of all intra-axonal movement, indicating that saltations of the two mobile structures are dependent on intra-axonal calcium. The predominant structure in varicosities is a large aggregation of an anastomosing, tubular, smooth endoplasmic reticulum embedded in an amorphous matrix, suggesting a form of "packaged" cytomembranes undergoing bulk transport. IPD inclusions, presumably carrying membranes, appear to shuttle between varicosities and growth cones during axon elongation, and between growth cones and varicosities during axon retraction. alpha-Spectrin, actin, and calmodulin were shown by immunocytochemistry to be preferentially distributed to varicosities and IPD inclusions. The co-transport of spectrin, actin, and calmodulin with cytomembranes undergoing rapid saltations departs from reported results of radioactive labeling experiments insofar as spectrin, actin, and calmodulin are not normally associated with rapidly transported membrane components in the latter studies. Possible reasons for the discrepancy are discussed. Our results suggest that spectrin and actin may play a role in the packaging and axoplasmic transport of cytomembranes concerned with plasmalemma recycling.

Actins↗

PubMed1983

Structure of human erythrocyte spectrin. II. The sequence of the alpha-I domain.

The complete sequence of 595 amino acids of the alpha-I domain of human erythrocyte spectrin has been determined. Peptides derived from three different protease cleavages were purified using high performance liquid chromatography and subjected to automated amino acid sequence analysis. These data along with sequences of the cyanogen bromide and large tryptic peptides (Speicher, D.W., Davis, G., Yurchenco, P.D., and Marchesi, V.T. (1983) J. Biol. Chem. 258, 14931-14937) represent most or all of the sequence of spectrin alpha-I. The single remaining ambiguity is the precise termination of the COOH terminus of the alpha-I domain. The sequence data suggest that the 595 residues presented here represent the complete sequence of the alpha-I domain, but the apparent size of the COOH-terminal CNBr fragment suggests the existence of an additional 38 residues at the end of the domain. The sequence of the alpha-I domain contains a single type of internal homology composed of multiple 106-amino acid repeats consistent with the occurrence of multiple gene duplications during the course of spectrin evolution. The only portion of the alpha-I sequence which does not appear to contain this sequence repeat is the segment containing the NH2-terminal 17 residues. This unique segment may be part of the oligomer binding site. No disulfide bonds appear to be involved in the structure of alpha-I and cysteine is not highly conserved. Calculations of secondary structure suggest the presence of short helices which fold into triple helical segments approximately 50 A in length. There is little beta sheet structure. A model of spectrin structure incorporating the repeat unit and proposed secondary structure is presented. A computer search of alpha-I sequence with the National Biomedical Research Foundation database of 2145 protein sequences did not detect any significant relationships. Spectrin is apparently the first member of a new class of proteins to be structurally characterized.

Amino Acid Sequence↗

PubMed1981

Anti-spectrin in sera containing smooth muscle autoantibodies from patients with chronic active hepatitis.

Sera from patients with chronic active hepatitis containing anti-smooth muscle autoantibodies (SMA) react against rabbit muscle actin as well as human red cell spectrin. These anti-spectrin antibodies recognize the same antigen as rabbit anti-human spectrin antibodies and are not involved in the staining pattern given by SMA-containing sera tested with smooth muscle sections. These anti-spectrin antibodies probably recognize an antigenic structure common to both spectrin and another as yet undetermined molecule (which however is not likely to be myosin).

Actins↗

PubMed1980

Structural characterization of the phosphorylation sites of human erythrocyte spectrin.

The phosphorylation sites of spectrin dimer were characterized before and after incubation of intact human red cells with [32P]orthophosphate. Phosphate measurements of unlabeled spectrin dimer show it contains 4.0 +/- 0.3 covalent protein phosphates, all located on band 2. Quantitation of the number of exchangeable phosphorylation sites in erythrocytes labeled with [32P]orthophosphate yields 3.9 +/- 0.3 phosphates/spectrin dimer, indicating that all four spectrin phosphorylation sites are metabolically active. Tryptic and chymotryptic peptide mapping reveals that the dimer contains three unique 32P-labeled tryptic peptides of approximately 4,600 (A1), 3,500 (A2), and 2,400 (B) daltons. Peptide A1 contains both phosphoserine and phosphothreonine while peptides A2 and B possess only phosphoserine. Peptides A1 and A2 remain associated after trypsinization of spectrin dimer and are only separable in detergents. All three 32P-labeled tryptic peptides are contained within a 20,000 dalton cyanogen bromide fragment which is within a 60,000 dalton staphylococcal protease phosphopeptide. All these fragments are found within a 90,000 dalton nitrothiocyanobenzoic acid phosphopeptide. The purified 20,000 dalton fragment contains no homoserine or homoserine lactone and is the COOH-terminal cyanogen bromide peptide of band 2. The isolated tryptic peptide B possesses no lysine or arginine and is presumably the COOH-terminal tryptic peptide of band 2 and the most distal phosphopeptide. Thus, the four phosphorylation sites of spectrin dimer are clustered at the extreme COOH-terminal end of band 2.

Adenosine Triphosphate↗

PubMed1994

Immunolocalization of calpain I-mediated spectrin degradation to vulnerable neurons in the ischemic gerbil brain.

Transient ischemia-induced perturbations in calcium homeostasis have been proposed to lead to pathological activation of the cysteine protease calpain I and subsequent delayed neuronal death in the CA1 region of hippocampus. We report here on the design and characterization of antibodies selective for calpain-generated fragments of brain spectrin, and their use for immunoblot and immunohistochemical analyses of calpain activation following cerebral ischemia in the gerbil. Although spectrin was susceptible to degradation in vitro by many mammalian proteases, only calpain degraded spectrin to generate fragments immunoreactive with the antibodies. Following 5 min of global ischemia, immunoreactivity for calpain-degraded spectrin was rapidly (within 30 min) and markedly elevated in the perikarya and dendrites of several populations of forebrain neurons. The rapid calpain activation was completely prevented by the NMDA receptor antagonist MK-801. At later times postischemia, but prior to frank neuronal necrosis, calpain-degraded spectrin was restricted to hippocampal area CA1 pyramidal neurons. Silver impregnation histochemistry confirmed that neuronal damage was confined to area CA1. The results indicate that while nonpathological NMDA receptor stimulation can activate calpain, only those neurons showing sustained calpain activation are destined to die.

Amino Acid Sequence↗

PubMed1993

Localization of dystrophin and beta-spectrin in vacuolar myopathies.

We examined the expression of the cytoskeletal proteins dystrophin and beta-spectrin on vacuolar boundaries in vacuolar myopathies. We also localized utrophin, a dystrophin homologue, and laminin, which served as a marker for the basal lamina. Four types of vacuoles were identified. Type 1 vacuoles, found in all diseases, were lined by laminin, dystrophin, and beta-spectrin and arose from infoldings of the basal lamina and sarcolemma into splitting or branching fibers. Type 2 vacuoles were lined by dystrophin and beta-spectrin and were most common in adult acid maltase deficiency, chloroquine myopathy, and periodic paralysis. Traces of utrophin were also noted on the boundaries of some type 2 vacuoles, but only in those fibers that also expressed utrophin on their surface membrane. Type 3 vacuoles were lined by small patches of dystrophin and beta-spectrin and occurred in any vacuolar myopathy. Type 4 vacuoles were unlined by any of the above antigens and were most common in infantile acid maltase deficiency and in the nonlysosomal glycogenoses. Immunoelectron microscopy confirmed the dystrophin label on vacuolar boundaries but revealed no reaction product on any other membranous component within the muscle fiber. We conclude that dystrophin and beta-spectrin provide cytoskeletal support for a species of membrane-bound vacuoles in diverse myopathies.

Dystrophin↗

PubMed1993

Functional characterization of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site.

Spectrin, a heterodimer composed of alpha and beta subunits, interacts with itself head-to-head to form tetramers in the erythrocyte membrane cytoskeleton. The NH2-terminal region of alpha-spectrin, encompassing the alpha I 80-kDa domain, was expressed in Escherichia coli. In addition to the correctly initiated polypeptide, four smaller polypeptides were produced by initiation at internal codons. Only the full-length polypeptide was able to bind to spectrin dimers, beta monomers, and to a recombinant polypeptide containing the COOH terminus of beta-spectrin. The head-to-head interaction with beta-spectrin was also retained by a recombinant polypeptide containing the NH2-terminal 158 amino acids of the alpha subunit. Deletion of the first 27 or 49 NH2-terminal amino acids abolished binding of this polypeptide to the beta monomer. The phasing used to design these recombinant polypeptides was based on a conformational model recently refined by Speicher et al. (Speicher, D. W., DeSilva, T. M., Speicher, K. D., Ursitti, J. A., Hembach, P., and Weglarz, L. (1993) J. Biol. Chem. 268, 4227-4235), where the structural unit begins and terminates around residue 30 of the repeat unit. The binding properties, mobility on gel filtration, and circular dichroism data of the recombinant polypeptides indicated that most polypeptides were able to assume their native conformation.

Amino Acid Sequence↗

PubMed1996

Expression of spectrin alpha I/65 hereditary elliptocytosis in patients from Brazil.

We report the clinical and laboratory findings in three unrelated families from southeastern Brazil with Sp alpha I/65 hereditary elliptocytosis (HE), including one homozygote and a patient presenting an elongated beta-spectrin. In family 1, three patients presented the allele alpha-Lely in trans to the elliptocytogenic allele. In these three patients the blood smear showed pronounced elliptocytosis, poikilocytosis and a few small red cell fragments instead of the mild elliptocytosis observed in their father, who did not present the polymorphism. In family 2 we describe one homozygote, with consanguineous parents presenting with anaemia, splenomegaly, severe poikilocytosis and elliptocytosis, budding, microspherocytes and numerous fragments in the blood smear. In family 3 we found an elongated beta Sp in a patient with Sp alpha I/65. The cause of the HE was the Sp alpha I/65 since the elongated beta Sp was not found in his brother, who also presented with HE and Sp alpha I/65. Apparently the abnormal beta Sp did not aggravate the HE, because both individuals had the same clinical and laboratory findings. However, the propositus presented a few more elliptocytes and poikilocytes than his brother, probably because the elongated beta-spectrin may have disturbed the spectrin self-association. In fact, in the propositus an abnormal band was observed in the nondenaturing gels, just above the Sp dimer, probably as a result of the association of the abnormal beta Sp with the normal spectrin chains. In the family studied here, both brothers presented the allele alpha Lely, but as their mother was dead, it was not possible to determine the polymorphism transmission. However, the high number of poikilocytes observed in the blood smear of both cases suggests an association in trans with the Sp alpha I/65. Thus, taken together, the data in this report indicate that HE secondary to Sp alpha I/65 abnormality is frequent in Brazil, and in one case it was associated with an apparently novel abnormal large beta-spectrin.

Adult↗

PubMed1996

Human erythroid spectrin alpha subunit and its SH3 domain are sensitive to acidic Plasmodium falciparum proteolytic activity.

Many proteases play a crucial role in the Plasmodium intraerythrocytic life cycle. Spectrin depletion, one of the major events involved in parasite release from the red blood cell, results from proteolytic activities associated with the presence of the intracellular parasite. Here, we describe a new acidic proteolytic activity from Plasmodium falciparum, whose target is the alpha-subunit of human spectrin. Immunoblotting experiments with antibodies specific for the tryptic peptides of the alpha-chain and in vitro proteolysis tests on recombinant peptides from different regions of the spectrin alpha subunit demonstrated that cleavage sites for the parasite proteolytic activity were localized within the SH3 motif of the alpha-chain sequence. Remarkably, this Plasmodium protease activity on spectrin SH3 substrate was unable to cleave the SH3 from fodrin, a non-erythroid spectrin.

Animals↗

PubMed1997

Biogenesis of surface domains in fly photoreceptor cells: fine-structural analysis of the plasma membrane and immunolocalization of Na+, K+ ATPase and alpha-spectrin during cell differentiation.

Electron microscopic examination of pupal and adult blowfly (Calliphora erythrocephala) retina provides novel details on the biogenesis of the photoreceptor surface, particularly regarding the development of the microvillar rhabdomere and associated structures, such as the submicrovillar endoplasmic reticulum. Localization of the Na+, K(+)-ATPase on the surface of developing photoreceptors has also been examined by immunofluorescence confocal microscopy and immunogold electron microscopy. Na+, K(+)-ATPase has a nonpolarized distribution in midpupal photoreceptors that are determined by fate but that are not yet completely differentiated. Large amounts of Na+, K(+)-ATPase are synthesized and delivered to the cell surface throughout the second half of pupal life. At certain time points in late pupal development, specific membrane domains become cleared of Na+, K(+)-ATPase in the photoreceptors R1-R6. However, the distribution of Na+, K(+)-ATPase remains nonpolarized in R7/R8, even after eclosion. Because the membrane-associated cytoskeleton plays a direct role in the establishment and maintenance of membrane domains in a variety of systems, it is of interest to study the distribution of alpha-spectrin and its possible association with Na+, K(+)-ATPase. The localization of alpha-spectrin resembles the distribution pattern of Na+, K(+)-ATPase in midpupal and adult photoreceptors. However, changes in Na+, K(+)-ATPase localization in late pupal photoreceptors precede the redistribution of alpha-spectrin by several days. Biochemical studies of cellular membranes demonstrate further that Na+, K(+)-ATPase can be solubilized by Triton X-100, although alpha-spectrin remains in a macromolecular complex. These results indicate that the development and the maintenance of the polarized Na+, K(+)-ATPase distribution in blowfly photoreceptors are not tightly coupled to alpha-spectrin.

Animals↗

PubMed1998

Spectrin localization in osteoclasts: immunocytochemistry, cloning, and partial sequencing.

The presence of spectrin was demonstrated in chick osteoclasts by Western blotting and light and electron microscopic immunolocalization. Additionally, screening of a chick osteoclast cDNA library revealed the presence of alpha-spectrin. Light microscope level immunocytochemical staining of osteoclasts in situ revealed spectrin staining throughout the cytoplasm with heavier staining found at the marrow-facing cell margin and around the nuclei. Confocal microscopy of isolated osteoclasts plated onto a glass substrate showed that spectrin encircled the organelle-rich cell center. Nuclei and cytoplasmic inclusions were also stained and the plasma membrane was stained in a nonuniform, patchy distribution corresponding to regions of apparent membrane ruffling. Ultracytochemical localization showed spectrin to be found at the plasma membrane and distributed throughout the cytoplasm with especially intense staining of the nuclear membrane and filaments within the nuclear compartment.

Animals↗

PubMed1999

Brain spectrin (fodrin) interacts with phospholipids as revealed by intrinsic fluorescence quenching and monolayer experiments.

We demonstrate that phospholipid vesicles affect the intrinsic fluorescence of isolated brain spectrin. In the present studies we tested the effects of vesicles prepared from phosphatidylcholine (PtdCho) alone, in addition to vesicles containing PtdCho mixed with other phospholipids [phosphatidylethanolamine (PtdEtn) and phosphatidylserine] as well as from total lipid mixture extracted from brain membrane. The largest effect was observed with PtdEtn/PtdCho (3:2 molar ratio) vesicles; the effect was markedly smaller when vesicles were prepared from egg yolk PtdCho alone. Brain spectrin injected into a subphase induced a substantial increase in the surface pressure of monolayers prepared from phospholipids. Results obtained with this technique indicated that the largest effect is again observed with monolayers prepared from a PtdEtn/PtdCho mixture. The greatest effect was observed when the monolayer contained 50-60% PtdEtn in a PtdEtn/PtdCho mixture. This interaction occurred at salt and pH optima close to physiological conditions (0.15 M NaCl, pH7.5). Experiments with isolated spectrin subunits indicated that the effect of the beta subunit on the monolayer surface pressure resembled that measured with the whole molecule. Similarly to erythrocyte spectrin-membrane interactions, brain spectrin interactions with PtdEtn/PtdCho monolayer were competitively inhibited by isolated erythrocyte ankyrin. This also suggests that the major phospholipid-binding site is located in the beta subunit and indicates the possible physiological significance of this interaction.

Animals↗

PubMed1996

Distribution of actin, myosin, and spectrin during enucleation in erythroid cells of hamster embryo.

Yolk-sac derived erythroblasts undergo semi-synchronous maturation and some of them enucleate in the peripheral blood of embryos. We have studied the assembly and distribution of actin, myosin, and spectrin during the enucleation of Syrian hamster embryonic erythroblasts. At day 11 of the gestation, that is just before the start of the enucleation, formation of a cytoskeletal structure consisted chiefly of particulate associations of F(filamentous)-actin was detected by the staining with rhodamine-labeled phalloidin. Stress-fiber-like structures were not observed in each differentiation stage after day 10. Distribution of myosin, actin, and spectrin was studied immunocytochemically to know the role of them in the enucleation of erythroid cells that starts at late day 11 or early day 12 in the gestation. The enucleation is preceded by the approach and the subsequent attachment of nucleus to the plasma membrane. At that time, actin and myosin are present in the cytoplasmic and cortical region of the cells. From the time when the extrusion of nucleus has started, condensation of actin and myosin was observed at the cell cortex area surrounding the extruding nucleus, and a contractile ring-like structure was infrequently observed. Spectrin was observed in the cortical region of the cells, and the change of the localization of spectrin was not observed throughout the terminal differentiation process (days 10-12) of the embryonic erythroid cells. The results show the possible involvement of a myosin-actin contractile system that appears around the extruding nucleus within the mechanism of erythroid enucleation.

Actins↗

PubMed1995

Actin, alpha-actinin, and spectrin with specific associations with the postacrosomal and acrosomal domains of bovine spermatozoa.

BACKGROUND: Characteristic membrane changes in spermatozoa culminating in acrosome reaction and sperm-egg fusion, and suspected involvement of actin-containing cytoskeleton in membrane changes in general, prompted us to investigate subcellular distribution of actin and actin-binding proteins in bovine spermatozoa subjected to various extractions which sequentially denude the sperm investments. METHODS: Spermatozoa were treated with either 1% SDS, 0.1% Triton X-100, 0.1% Hyamine, or 1 M MgCl2 or were sonicated. Immunostaining of actin, alpha-actinin, spectrin, and acrosin as well as electron microscopic analysis of extracted spermatozoa were carried out. RESULTS: Extractions caused evagination of the acrosomal lamina which retained focal contacts with the inner acrosomal membrane. Extractions further revealed lateral prongs at the anterior border of the postacrosomal sheath. Labeling for alpha-actinin and spectrin was localized in the acrosin-positive acrosomal lamina, neck, and principal piece, the latter containing also relatively extraction-resistant oligomeric or polymerized actin. In the postacrosomal area, actin was accumulated in the extraction-resistant posterior ring structure and anteriorly at the sites apparently related to the lateral prongs. Notably, spectrin reactivity was enhanced by MgCl2 in head, neck, and principal piece, and sonication abolished cytoskeletal immuno-reactivity in the head. CONCLUSIONS: Destabilization of membranes with selected extractions induces changes in the acrosomal lamina mimicking acrosomal vesicle formation. The lateral prongs and posterior ring structure, respectively, may serve as anterior and posterior anchors for the extraction-resistant post-acrosomal sheath. The lateral prongs may also be a merger zone for actin, alpha-actinin, and spectrin with important implication on sperm function. The latter two proteins may be involved in acrosomal vesicle formation. It is apparent that extractions have a significant effect on the detectability of sperm cytoskeletal elements.

Acrosome↗

PubMed1982

Spectrin domains: proteolytic susceptibility as a probe of protein structure.

Mild treatment of human erythrocyte spectrin with trypsin produces discrete intermediate-sized peptides. The effects of buffer composition, enzyme-substrate ratio, temperature, and other experimental parameters on the resulting peptide pattern have been examined. Spectrin is capable of regaining its proteolytic resistance after NaDodSO4-induced denaturation, permitting the use of isolated subunits to study spectrin structure and function. Tryptic digestion of isolated subunits also has greatly facilitated the identification of the subunit origin of the intermediate-sized peptides. Isolated subunits could also be recombined to form functional units similar but not identical to the native dimeric form of the regions or domains connected by small protease sensitive segments. The structural integrity and accessibility of these sites is minimally affected by oligomeric state or proteolytic digestion conditions. The similarities of sizes, isoelectric points, and amino acid compositions of many intermediate-size peptides from areas of both subunits suggest that at least part of spectrin's structure may have evolved via replication of a single gene. A possible structural repeat of approximately 50,000 daltons is hypothesized.

Chemical Phenomena↗