Molecular weight of major component proteins in crude saline extract of adult Paragonimus westermani

When the component proteins in crude saline extract of 13-week old adult Paragonimus westermani were observed by non-denaturing discontinuous-polyacrylamide gel electrophoresis (Disc-PAGE), 8 distinct bands were clearly recognized. Molecular weight (MW) of each band protein, numbered in sequence from cathodal side which appeared in 10% separating gel, was measured first by Ferguson plot utilizing different gel concentrations from 10% to 4.5%. MW of band 1 protein (known as egg protein) was 440 kDa. And MW of other band proteins were: 386 kDa in band 2, 17.4 kDa in band 3, 17 kDa in band 4, 14.3 kDa in band 5, 46 kDa in band 6, 38 kDa in band 7 and 23 kDa in band 8. When the proteins in the crude extract were separated into fractions by molecular sieve chromatography through 1.6(phi) X 70 cm sized Sephacryl S-300 Superfine column and revisualized by Disc-PAGE in 8% gel, the sequence of eluted proteins was band 1, band 2, band 6, band 7 and bands 3, 4, 5 and 8. This elution profile confirmed MW of each band protein in the crude extract as measured by Ferguson plot.     

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.     

Modulation of erythrocyte band 4.1 binding by volume expansion

Erythrocyte band 4.1 is an important protein in the control and maintenance of the cytoskeleton. Skate erythrocyte band 3, the anion exchanger, appears to play a pivotal role in the regulation of volume-stimulated solute efflux during volume expansion. Because band 4.1 interacts with band 3, we tested whether their interaction might change during volume expansion. Skate red blood cells were volume-expanded in either hypotonic media (one-half osmolarity) or were swollen under isoosmotic conditions by inclusion of ethylene glycol or ammonium chloride in the medium. Microsomal membranes isolated from red cells under volume expanded conditions demonstrated a significant decrease in the amount of band 4.1 bound to band 3. In unstimulated cells, approximately one third of the binding of band 4.1 occurred to band 3. This binding was characterized as being sensitive to competition by the peptide IRRRY. The majority of band 4.1 is bound to glycophorin (as demonstrated in other species), and this binding does not change during volume expansion. The alteration in band 4.1:band 3 interaction occurs within 5 min after volume expansion and is transient, returning to near normal interaction within 60 min. Two drugs that promote band 3 oligomerization, pyridoxal-5'-phosphate and DIDS, also decreased band 4.1 interaction with band 3. Band 4.1 and ankyrin binding to band 3 may be reciprocally related as high-affinity ankyrin binding sites to band 3 observed under volume-expanded conditions are decreased by inclusion of band 4.1 in the binding reactions. J. Exp. Zool. 289:177-183, 2001.     

Molecular basis and functional consequences of the dominant effects of the mutant band 3 on the structure of normal band 3 in Southeast Asian ovalocytosis

Southeast Asian ovalocytosis (SAO) human red cell membranes contain similar proportions of normal band 3 and a mutant band 3 with a nine amino acid deletion (band 3 SAO). We employed specific chemical modification and proteolytic cleavage to probe the structures of band 3 in normal and SAO membranes. When the membranes were modified specifically at lysine residues with N-hydroxysulfosuccinimide-SS-biotin, band 3 Lys-851 was not modified in normal membranes but quantitatively modified in SAO membranes. Normal and SAO membranes showed different patterns of band 3 proteolytic cleavage. Notably, many sites cleaved in normal membranes were not cleaved in SAO membranes, despite the presence of normal band 3 in these membranes. The mutant band 3 changes the structure of essentially all the normal band 3 present in the SAO membranes, and these changes extend throughout the normal band 3 molecules. The results also imply that band 3 in SAO membranes is present as hetero-tetramers or higher hetero-oligomers. The dominant structural effects of band 3 SAO on the other band 3 allele have important consequences on the functional and hematological properties of human red cells heterozygous for band 3 SAO. Analysis of the altered profile of biotinylation and protease cleavage sites suggests the location of exposed surfaces in the band 3 membrane domain and identifies likely interacting regions within the molecule. Our approach provides a sensitive method for studying structural changes in polytopic membrane proteins.     

Alcune caratteristiche strutturali dei cromosomi politenici del sospensore embrionale di Phaseolus coccineus in due momenti dell'embriogenesi

Abstract

Some features of polytene chromosomes of Phaseolus coccineus suspensor during two stages of early embryogenesis. – The distribution of DNA and RNA puffs in the whole genome of the giant cells of the Phaseolus coccineus embryo suspensor has been detected in two stages of embryo development. The collected data show that the chromosome regions showing the highest frequency of DNA puffs in both analysed stages are the following: i) band B (the fraction proximal to secondary constriction) of chromosome pair I and band E of chromosome pair V. When the two stages of development are however compared, it is seen that the % of DNA puffs in chromosome pair is at least double in suspensors dissected from the first stage of embryo development (86% in the first stage; 41% in the second stage). As to chromosome pair V band E organizes DNA puffs in 36% and in 50% of observed chromosomes in the first and second stage respectively; ii) band A of chromosome pair II, with a frequency of 52% (first stage) and 27% (second stage); iii) band E of chromosome pair VIII (27% in the first stage and 19% in the second stage). As far as the organization of RNA puffs is concerning it seems possible to outline the following values as the highest percentages:

a) First stage. chromosome b) Second stage. chromosome

I: band B 83% I: band B 91% VI: band E 55%

II: band A 70% II: band A 51% band G 50%

IV: band E 45% band C+D 59% VIII: band E 44%

V: band B+C 57% IV: band E 51% IX: band A 54%

band E 71% band G 51% band E 43%

VI: band E 53% band I 51% band F 53%

VIII: band E 43% V: band E 43%

IX: band E 42%

The differences observed between the two stages are discussed in relation to the function of the suspensor.     

Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes.

The effects of human red cell glycophorin A (GPA) on the expression of the human erythrocyte anion transporter (band 3, AE1) has been examined in Xenopus oocytes. The coexpression of GPA with band 3 increased stilbene disulfonate-sensitive chloride transport into the oocytes. The effect of GPA was particularly noticeable at low band 3 concentrations and less marked at high band 3 cRNA concentrations. The enhancement of chloride transport was specific to GPA and was not observed when either glycophorin B or glycophorin C was coexpressed with band 3. Immunoprecipitations of whole oocyte homogenates showed the amount of band 3 synthesized was not affected by GPA at subsaturating cRNA concentrations. More band 3 was detected at the oocyte surface by immunoprecipitation when GPA was also expressed. Chymotrypsin treatment of intact oocytes was also used to assess surface band 3 and greater cleavage of band 3 by chymotrypsin was observed when GPA was present. Band 3 synthesis and assembly into canine pancreatic microsomes in the reticulocyte cell-free translation system was not altered by cotranslation of GPA. We suggest that GPA facilitates the translocation of band 3 to the plasma membrane at some point during band 3 biosynthesis in Xenopus oocytes. However, GPA is not essential for the expression of band 3 in red cells, since GPA-deficient individuals have apparently normal levels of band 3. Other GPA-independent mechanisms must also allow translocation of band 3 to the surface membrane in erythroid cells and oocytes. GPA may affect the rate of accumulation of band 3 at the cell surface, rather than the final level in the plasma membrane.     

Multicolor Electroluminescence from Intermediate Band Solar Cell Structures

Intermediate band solar cells are a new generation of photovoltaics that allow for better utilization of the solar spectrum. The key and most challenging requirement for these cells is an efficient optical coupling between the intermediate band and the charge conducting bands. GaNAs based intermediate band solar cells have been used to generate electroluminescence. Two electroluminescence peaks are generated in the structure with electrically blocked intermediate band. The peaks are observed for both forward and reverse bias configuration and are attributed to optical transitions from the conduction to the intermediate band, and from the intermediate band to the valence band. The origin of the electroluminescence is confirmed by the temperature dependence of the electroluminescence peak energies that is consistent with the band anticrossing model of the intermediate band formation in dilute nitride alloys. This is the first direct observation of the optical transitions required for the operation of intermediate band solar cells. The results also demonstrate that properly modified intermediate band solar cell structures could be used as multicolor light emitters.     

Localization of projectin in locust flight muscle

Projectin is a giant filamentous protein of arthropod striated muscle. By using immunofluorescence microscopy, projectin was shown to span between the I band and the A band in locust (Locusta migratoria) flight muscle sarcomeres. The N- and C-terminal regions of projectin molecules were localized in the I band and A band, respectively. This observation explains the controversial reports of previous studies that projectin is localized either in the I band or in the A band of locust flight muscle sarcomeres. It is also observed that the N-terminal region of projectin is located in the I band of locust leg muscle sarcomeres.     

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.     

Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3.

Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.     

Role ofN-Myristylation in Targeting of Band 4.2 (Pallidin) in Nonerythroid Cells

Band 4.2 (pallidin) is a major erythrocyte membrane protein which has been detected in a number of nonerythroid cell types. Increasing evidence suggests that band 4.2 is involved in maintaining membrane stability in the erythrocyte. For example, band 4.2 binds to the integral membrane protein band 3 and to cytoskeletal proteins in the erythrocyte membrane, and band 4.2 deficiency results in varying degrees of hemolytic anemia. We have previously shown that human erythrocyte band 4.2 is myristylated at its penultimate glycine. Here we report that when expressed in both Sf9 and COS cells, myristylated forms of band 4.2 are detected at different intracellular locations than nonmyristylated forms. We also show that the unspliced form of human erythrocyte band 4.2 (a minor form in reticulocytes which contains an additional 30 amino acids after the first three N-terminal amino acids compared to the major erythroid form) is myristylated only at a barely detectable level, while mouse erythrocyte band 4.2 (homologous to the major erythroid form of human band 4.2) is myristylated at a level comparable to that of human band 4.2. These results suggest that myristylation plays a key role in the targeting of band 4.2 to specific intracellular locations and is likely to have a role in the function of this protein.     

Structural organisation of band 3 in Melanesian ovalocytes

The diffusional freedom of human erythrocyte band 3 (anion exchanger 1) has been measured in membranes from normacytic and ovalocytic erythrocytes. A dramatic reorganisation of band 3 in the ovalocyte membranes is indicated by a markedly restricted rotational mobility. Extraction of spectrin from erythrocyte membranes had no effect on normocyte band 3 mobility, but partially relieved the restrictions on ovalocyte band 3 mobility. Further removal of ankyrin and band 4.2 resulted in an increase in the rotational mobility of both ovalocyte and normocyte band 3 to similar levels. The results suggest that the molecular basis of the unusual shape and decreased deformability of ovalocytes resides in an altered interaction of band 3 with one or more of the peripheral proteins. We present a model which illustrates a possible role for band 3 aggregation in controlling erythrocyte deformability.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Red cell membrane protein band 4.2: phenotypic,genetic and electron microscopic aspects

The present status of band 4.2 has been reviewed from the standpoint of protein chemistry, gene analysis and clinical hematology.Band 4.2 plays an important role in various cellular functions. In 142 GCT → ACT band 4.2 deficiency, abnormalities of the cytoskeletal network were clearly observed by electron microscopy and by ektacytometry, although the cytoskeletal proteins themselves were essentially normal in these red cells. The physiological states of band 3 in situ in the membranes were also affected in band 4.2 deficiency, as detected by electron microscopy, although again the biochemical properties of band 3 itself were essentially normal in these red cells.Other disorders of band 4.2 deficiency in the absence of the 142 GCT → ACT mutation appear to be most interesting in the pathogeneesis of hemolysis.In some of the band 4.2 anomalies, other membrane proteins including band 3 would appear to be most pathognomonic for the disease states. These conditions require elucidation by protein chemistry and gene analysis.The control mechanism of the gene expression should also be clarified to understand the important role of band 4.2 in health and disease.     

The complex of band 3 protein of the human erythrocyte membrane and glyceraldehyde-3-phosphate dehydrogenase: stoichiometry and competition by aldolase

The cytoplasmic domain of band 3, the main intrinsic protein of the erythrocyte membrane, possesses binding sites for a variety of other proteins of the membrane and the cytoplasm, including the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase. We have studied the stoichiometry of the complexes of human band 3 protein and GAPDH and the competition by aldolase for the binding sites. In addition, we have tried to verify the existence of mixed band 3/GAPDH/aldolase complexes, which could represent the nucleus of a putative glycolytic multienzyme complex on the erythrocyte membrane. The technique applied was analytical ultracentrifugation, in particular sedimentation equilibrium analysis, on mixtures of detergent-solubilized band 3 and dye-labelled GAPDH, in part of the experiments supplemented by aldolase. The results obtained were analogous to those reported for the binding of hemoglobin, aldolase and band 4.1 to band 3: (1) the predominant or even sole band 3 oligomer forming the binding site is the tetramer. (2) The band 3 tetramer can bind up to four tetramers of GAPDH. (3) The band 3/GAPDH complexes are unstable. (4) Artificially stabilized band 3 dimers also represent GAPDH binding sites. In addition it was found that aldolase competes with GAPDH for binding to the band 3 tetramer, and that ternary complexes of band 3 tetramers, GAPDH and aldolase do exist.     

A thermoluminescence study of the effects of nitrite on photosystem II in spinach thylakoids.

The site of action of nitrite on PS II was investigated by measuring the TL profile of nitrite-treated spinach thylakoid membranes. Three bands were observed in control, which were identified as the Q band (7 degrees C), the B band (24 degrees C) and the C band (57 degrees C). In the presence of 20 mmol/L nitrite, the intensity of the Q band decreased, the B band upshifted to 46 degrees C but the C band disappeared. The suppression of the Q band and the upshift of the B band suggested that nitrite caused inhibition at the water oxidizing complex. The effects of nitrite also remained the same in the presence of chloride. In case of ion-sufficient thylakoid membranes, nitrite decreased the Q band peak intensity and caused an upshift in the B band peak temperature. Nitrite showed similar effects in the presence of DCMU. This suggested that the site of action of nitrite is not at the acceptor side but at the donor side of PS II. The inhibition shown by nitrite has been found to be specific for nitrite anion. No other anions such as formate, fluoride or nitrate, were effective.     

Both ankyrin and band 4.1 are required to restrict the rotational mobility of band 3 in the human erythrocyte membrane.

A population of band 3 proteins in the human erythrocyte membrane is known to have restricted rotational mobility due to interaction with cytoskeletal proteins. We have further investigated the cause of this restriction by measuring the effects on band 3 rotational mobility of rebinding ankyrin and band 4.1 to ghosts stripped of these proteins as well as spectrin and actin. Rebinding either ankyrin or 4.1 alone has no detectable effect on band 3 mobility. Rebinding both these proteins together does, however, reimpose a restriction on band 3 rotation. The effect on band 3 rotational mobility of rebinding ankyrin and 4.1 are similar irrespective of whether or not band 4.2 is removed from the membrane. We suggest that ankyrin and 4.1 together promote the formation of slowly rotating clusters of band 3.     

Golgi secretion is not required for marking the preprophase band site in cultured tobacco cells

The preprophase band predicts the future cell division site. However, the mechanism of how a transient preprophase band fulfils this function is unknown. We have investigated the possibility that Golgi secretion might be involved in marking the preprophase band site. Observations on living BY-2 cells labeled for microtubules and Golgi stacks indicated an increased Golgi stack frequency at the preprophase band site. However, inhibition of Golgi secretion by brefeldin A during preprophase band formation did not prevent accurate phragmoplast fusion, and subsequent cell plate formation, at the preprophase band site. The results show that Golgi secretion does not mark the preprophase band site and thus does not play an active role in determination of the cell division site.