Gels Of Membranes Research Articles

Quantitative analysis of the erythrocyte membrane proteins in polycythemia vera patients treated with hydroxycarbamide

More than 90% of polycythemia vera (PV) patients have a mutation in the protein JAK2, which is closely associated with the erythrocyte membrane. With the comparison of 1-D gels of erythrocyte membranes obtained from PV patients treated with hydroxycarbamide and those of untreated controls we observed significant differences in the region of 40–55kDa. On the basis of the LC–MS/MS analysis of this region we report up-regulation of four protein disulfide isomerases, which was subsequently confirmed by targeted mass spectrometric analysis. In further studies it will be prudent to compare this in patients both treated and not treated with hydroxycarbamide.

Study on structural evolution of perfluorosulfonic ionomer from concentrated DMF-based solution to membranes

To investigate the solution-cast process from concentrated DMF-based perfluorosulfonic ionomer (PFSI) solution to membranes and morphology of the corresponding solution-cast membranes, XRD measurements were conducted on concentrated solutions, gels of various ionomer concentrations and membranes prepared at different temperatures, and small-angle X-ray scattering from the membranes was studied in order to investigate their internal supermolecular structure. It is with interaction of backbones of the ionomer molecules that the PFSI aggregates in the solutions fuse into each other to form gels and membranes, with capillary liquid bridge force and thermal movement of the polymer chains as the driving force on drying. Fusion degree of the ionomer aggregates increases with increasing casting temperature. Accordingly, proton conductivity, water uptake, solubility in ethanol/water mixture and crystallinity of the membranes decrease with increasing casting temperature, whereas dry density and tensile stress at break of the solution-cast membranes increase. SEM and TEM measurements were also conducted to give direct observation of the structure of the membranes. It was also found that morphology of DMF-based solution-cast PFSI membranes is greatly different from that of Nafion ® thermo-extruded perfluorosulfonic membranes.

Proteomic analysis of the oxygen-evolving complex of photosystem II under biotec stress: Studies on Nicotiana benthamiana infected with tobamoviruses.

We have previously shown that tobamovirus infection induces an inhibition of photosystem II electron transport, disturbing the oxygen-evolving complex (OEC). In the infected plants, the OEC polypeptide pattern was modified when compared to healthy plants, the levels of the PsbP and PsbQ extrinsic proteins being lowered to different extents. In this work we have further investigated by two-dimensional polyacrylamide gel electrophoresis (2-DE) the changes on the OEC protein pattern of thylakoid membranes isolated from Nicotiana benthamiana Domin plants infected with the Spanish strain of pepper mild mottle virus. When the thylakoid membranes from healthy plants were analyzed for the presence of PsbO and PsbP proteins by 2-DE (pI range 4-7) and further immunoassayed by using specific-antisera against these two proteins, it was observed that four polypeptides cross-reacted with each antiserum. These data, along with the N-terminal amino acid sequence determined for the eight polypeptides, indicate that the N. benthamiana PsbO and PsbP proteins correspond to protein families. In the silver-stained 2-DE gels of thylakoid membranes isolated at different days postinoculation from virus-infected plants, it was observed that the content of PsbP polypeptides decreased dramatically with respect to those of PsbO, during the progress of the infection. Interestingly, there was a differential decrease of the different PsbP proteins, indicative of a distinct regulation of their expression.

How the Chlorophyll-Proteins got their Names

This is the story of how we started studying the green bands seen on SDS-polyacrylamide gels of thylakoid membranes dissociated with the non-ionic detergent beta-octyl-D-glucopyranoside. We explain some of the complications we and other workers encountered along the pathway to untangling the chlorophyll-protein complexes of higher plants, and give a concise summary of the complexes, their polypeptides and their genes.

Alterations in thylakoid membrane composition induced by iron starvation in sunflower plants

Previous analysis of photosynthetic electron transport of isolated thylakoid membranes in iron‐deficient sunflower plants showed a decrease in the whole chain and photosystem I (PSI) activities, whereas photosystem II (PSII) activity resulted to be unaffected (Tognini et al., 1996). On the basis of these results, the analysis of the molecular composition of thylakoid membranes was carried out. By using immuno‐blotting methods, a decrease in the protein content of PSI in iron‐deficient plants was detected, while light‐harvesting complex II (LHCII) increased and PSII core complex (CC) did not change compared to the controls. In iron‐deficient plants, the tetramethylbenzidine staining of thylakoid protein gels showed a decrease in the content of cytochrome f and cytochrome b6, while cytochrome bS59 remained unchanged in control and stressed plants. Green gels of thylakoid membranes seemed to confirm the above results; the green band corresponding to PSI holocomplex resulted less intense in chlorotic samples and the green bands of the lowest molecular weight, corresponding to LHCII, were increased in iron‐deficient thylakoid membranes in comparison to the controls. The HPLC analysis of the pigments associated with the green bands separated by Deriphat PAGE showed that the xanthophylls associated with both photosystems shifted toward the de‐epoxidated form following iron starvation, indicating the onset of a protective mechanism against photoinhibition at the levels of both photosystems.

Photosynthetic light-harvesting function of carotenoids in higher-plant leaves exposed to high light irradiances

Exposure of barley (Hordeum vulgare L.) leaves to strong white light (1500 μmol photons · m−2 · s−1) decreased the quantum yield of photosynthetic oxygen evolution in green light preferentially absorbed by carotenoids (Φ-510) but not in red light exclusively absorbed by chlorophylls (Φ-650). This phenomenon was observed to be (i) rapidly induced (within a few minutes), (ii) slowly reversible in darkness (within about 1 h), (iii) insensitive to dithiothreitol and (iv) maximally induced by photon flux densities higher than about 1000 μmol · m−2 · s−1. Determination of the carotenoid composition of the major light-harvesting complex of PSII (LHCII) and analysis of the thylakoid membrane lipid fluidity before and after strong illumination of barley leaves in the presence or the absence of dithiothreitol showed that the light-induced decrease in the Φ-510/Φ-650 ratio did not require the physical detachment of carotenoids from the pigment antennae. Compared to barley plants grown under moderate light and temperature conditions, plants grown in sustained high irradiance at elevated temperature exhibited (i) a lower Φ-510/Φ-650 ratio, (ii) a reduced size of the functional PSII pigment antenna in green light (but not in red light) and (iii) a marked increase in the amount of free carotenoids found in non-denaturing Deriphat-containing electrophoretic gels of thylakoid membranes. Similarly, the Φ-510/Φ-650 ratio of the LHCII-deficient chlorina-f2 barley mutant was very low compared to the wild type. Separation and quantification of the cis/trans carotenoid isomers of barley leaves revealed that strong illumination did not induce pronounced cis-trans isomerization of xanthophylls. Taken together, the data suggest that the efficiency of energy transfer from carotenoids to chlorophylls varies with the light environment both in the short term and in the long term, with excess light energy noticeably inhibiting the photosynthetic light-harvesting function of carotenoids. The photoprotective significance of this carotenoid decoupling from the chlorophyll antennae is discussed.

Changes in thylakoid protein patterns and antioxidant levels in two wheat cultivars with different sensitivity to sulfur dioxide

Plants of two wheat cultivars ( Triticum aestivum L., cultivars ‘Mec’ and ‘Chiarano’), each with a different sensitivity to sulfur dioxide (SO 2), were exposed continuously to 2, 23, 64 and 96 nl l −1 SO 2 for 4 months. To investigate whether the higher resistance to SO 2 of Chiarano was due to increased activities of detoxification systems, some antioxidant enzymatic and non-enzymatic systems were analyzed. Moreover, to test whether the different responses of the two cultivars to SO 2 were related to different changes at thylakoid level, we analyzed the thylakoid polypeptide patterns by two-dimensional electrophoresis. Two-dimensional gels of thylakoid membranes revealed a more pronounced generalized decrease of spot optical density in the more SO 2-sensitive Mec; following SO 2 fumigation, changes were observed in the area with molecular masses 26–29 kDa and isoelectric point (pI) 5–6 and molecular masses 24–27 kDa and pI 5.7–5.8. A significant decrease in optical density of a spot with a molecular mass of 32 kDa and pI 5.4, reported to be D1 protein of the photosystem II reaction centre, was observed in the leaves of both cultivars fumigated with 96 nl l −1 SO 2. Catalase activity was unaffected by SO 2 exposure. Following SO 2 fumigation, superoxide dismutase activity decreased in Chiarano, but remained unchanged in Mec. Both cultivars were found to increase guaiacol and ascorbic peroxidase activities as a response to SO 2. The redox status of ascorbic acid was similar in both cultivars and was unaffected by SO 2. However, the content of ascorbic acid was higher in the more tolerant Chiarano than in Mec at all SO 2 concentrations. The data reported here confirmed the different sensitivities to SO 2 of the two cultivars, as demonstrated by thylakoid protein analysis, and suggest that this difference depends on a differential ability to maintain elevated levels of ascorbic acid rather than on increasing detoxifying enzyme activities.

Low-Protein Diet Regulates a Proximal Nephron Insulin-like Growth Factor Binding Protein

Previous studies have demonstrated that the glomerulus and proximal tubule basolateral membrane possess both insulin-like growth factor (IGF) receptors and IGF-binding proteins (IGFBPs). The purpose of this study is to examine the control of these proteins in defined nephron segments during dietary protein restriction. Animals were fed isocaloric 6% (low-protein [LP]) or 40% (high-protein) protein diets for 7 to 10 days. 125I[IGF] affinity labeling of membranes prepared from isolated glomeruli or proximal tubule basolateral membranes demonstrated two proteins on autoradiograms of 6% polyacrylamide gels with molecular weights of 140,000 d and more than 200,000 d that were blocked by 300 nmol/L unlabeled IGF-I. The lower molecular weight species has been identified previously as the alpha subunit of the IGF-I receptor and was upregulated by a 6% LP diet. The upregulation of the IGF-I receptor was evident on 12% polyacrylamide gels of both glomeruli and basolateral membranes. Another protein with a molecular weight of approximately 38,000 d also was upregulated by LP diet. The protein was evident on 125I-IGF-I ligand blots and was immunostained with IGFBP-5 antibodies. Cytosol prepared from cortical tissue also demonstrated a 31,000-d protein that was immunostained with IGFBP-5 antibodies in animals fed a LP diet, but not in animals fed a high-protein or normal diet. RNA prepared from cortical tissue and hybridized with a IGFBP-5 cDNA probe revealed a 6.0-Kb transcript that was increase by LP diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease

The immunoglobulin A1 (IgA1) proteases of Neisseria gonorrhoeae have been defined as having human IgA1 as their single permissive substrate. However, in recent years there have been reports of other proteins which are susceptible to the proteolytic activity of these enzymes. To examine the possibility that gonococcal membrane proteins are potential substrates for these enzymes, isolated outer and cytoplasmic membranes of N. gonorrhoeae were treated in vitro with exogenous pure IgA1 protease. Analysis of silver-stained sodium dodecyl sulfate-polyacrylamide gels of outer membranes indicated that there were two outer membrane proteins of 78 and 68 kDa which were cleaved by IgA1 protease in vitro in GCM 740 (a wild-type strain) and in two isogenic IgA1 protease-negative variants. Similar results were observed with a second gonococcal strain, F62, and its isogenic IgA1 protease-negative derivative. When GCM 740 cytoplasmic membranes were treated with protease, three minor proteins of 24.5, 23.5, and 21.5 kDa were cleaved. In addition, when outer membranes of Escherichia coli DH1 were treated with IgA1 protease, several proteins were hydrolyzed. While the identities of all of these proteolyzed proteins are unknown, the data presented indicate that there are several proteins found in the isolated membranes of gram-negative bacteria which are permissive in vitro substrates for gonococcal IgA1 protease.

Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species

The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus.

The Leukocyte Common Antigen Family

The leukocyte-common antigen (L-CA) family is a group of high molecular weight glycoproteins uniquely expressed on the surface of all leukocytes and their hemopoietic progenitors ( 114). Members of this family differ by both protein sequence and carbohydrate structures and are expressed by leukocyte populations in specific patterns ( 15-27). An example of the differential expression is shown for the rat L-CA family in Figure 1 ( 19). Thymocytes express the lowest apparent molecular weight form of 1 80 kd; B lymphocytes express the highest form, 220 kd; and T lymphocytes express multiple forms. Differences also exist between T-cell subsets (Figure 1 C); CD8 T cells (Te/s) express the higher molecular weight forms more abundantly than do CD4 T cells (T H) ' The cell-type-specific patterns of expression are conserved throughout mammalian evolution ( 1, 2, 91 1, 19, 28), and there appear to be similar patterns of expression in chicken lymphocytes (29). L-CA is referred to in the literature by different names, including T200 (30), B220 for the B cell form ( 1 2) , the mouse allotypic marker Ly-5 ( 3 1 ) and more recently CD45 (32). L-CA is the most accurate descriptive name and is used for the purpose of this review. The L-CA family is a major cell surface component of lymphocytes and carries much of the carbohydrate of these cells. It has been estimated that 10% of the lymphocyte surface is occupied by one or more L-CA members (33). Because of this abundance, L-CA was easily detected on SDS-poly­ acrylamide gels of lymphocyte membranes (36-39) (Figure lA). It was also initially characterized as the major specificity of antilymphocyte sera (34) and as an allotypic marker (31 , 35). The primary protein structure has been determined from the analysis of cDNA clones, and this information,

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1.

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.

The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12.

A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide.

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis.

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.

Solubilization of Plant Membrane Proteins for Analysis by Two-Dimensional Gel Electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.

Protein alterations in Manduca sexta midgut and haemolymph following treatment with a sublethal dose of Bacillus thuringiensis crystal endotoxin

A single sublethal dose of Bacillus thuringiensis crystal toxin was delivered to fifth instar larvae of Manduca sexta to examine the effect of the toxin on the peritrophic membrane, midgut, and haemolymph. Twelve major peptide bands, ranging in size from above 200 kd down to 24 kd, were seen in silver-stained polyacrylamide gels of control peritrophic membranes. Twenty-four hours after toxin treatment four major glycoproteins were much reduced or absent from the peritrophic membranes. Concurrently, a new 32 kd protein (PM10) appears in the peritrophic membrane. This protein is still present 72 hr after toxin treatment when the peritrophic membranes' glycoproteins have returned to control levels, and the insect has apparently recovered from the toxin. A comparison of gels of the soluble midgut proteins revealed several bands which are common to both the midgut and the peritrophic membranes. Midgut contents taken from larvae 24 hr after toxin treatment had a large number of bands which were not present in the midgut contents of control larvae. Although the protein profile of haemolymph taken from larvae 24 hr following toxin treatment was apparently unaltered from control haemolymph, 72 hr following treatment a protein band, designated M13, was induced. M13 is associated with the immune response in M. sexta. This is the first report that the proteins associated with the immune response are induced by something other than a bacterial injection.

Insulin-stimulated phosphorylation and insulin binding in canine renal basolateral membranes.

To characterize the interaction of insulin with the renal proximal tubular cell, we measured insulin-stimulated phosphorylation in basolateral membranes and brush border membranes isolated from canine renal cortex. Insulin stimulated the specific phosphorylation of a 92,000 Mr protein band demonstrable on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of basolateral membranes. Dephosphorylation of the 92,000 Mr band occurred over time. Insulin-stimulated phosphorylation was concentration dependent, being clearly detectable at 10(-9) M insulin and maximal at 10(-6) M. The phosphorylated 92,000 Mr protein band from detergent-solubilized basolateral membranes was immunoprecipitated using serum from a patient with anti-receptor antibodies. No insulin-stimulated phosphorylation was detected in brush border membranes. Binding of insulin to membranes was highly specific for native hormone and was severalfold greater in basolateral membranes than in brush border membranes. These observations are consistent with the asymmetrical distribution of insulin-stimulated protein kinase as well as specific insulin binding sites in the proximal tubular cell. The data suggest that insulin exerts physiological effects on the cell through binding to specific basolateral membrane receptors and phosphorylation of those receptors.

Boar sperm cytoplasmic droplets: Their ultrastructure, their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes

Cytoplasmic droplets of the boar are progressively lost from the flagellum of boar spermatozoa during epididymal transit, at ejaculation and during the nitrogen cavitation technique for isolation of plasma membranes. Apparently very fragile, these structures are broken up in the fluids of the reproductive tract and in the buffer used during the nitrogen cavitation procedure. The maximal potential contamination of cytoplasmic droplet internal vesicular membranes in plasma membrane fractions was determined to be 2.2% of the entire membrane surface area collected. The highly sensitive silver-stained, two-dimensional (2-D) polyacrylamide (PAGE) gels of boar sperm plasma membranes did not reveal cytoplasmic droplet, internal membrane, marker polypeptides, further demonstrating the high purity of plasma membrane preparations. In addition, freeze-fracture demonstrates that the internal membranes of the cytoplasmic droplet show few intramembranous particles and these may contribute little protein to plasma membrane preparations. The presence of two forms of vesicular elements in boar sperm Cytoplasmic droplets (typical vesicles and collapsed vesicles) is described.

Isolation and characterization of phagosomes containing Chlamydia psittaci from L cells.

The obligate intracellular procaryote Chlamydia psittaci enters host cells by a mechanism similar to, but distinct from, conventional phagocytosis. To better understand chlamydial uptake, L-cell phagosomes containing a single chlamydial cell were isolated and studied. Two rounds of dextran rate-zonal gradient centrifugation of L cells homogenized 1 h after infection with C. psittaci yielded phagosomes relatively free of other membranous structures. In double-label experiments, the phagosomes were enriched over 40-fold for radioactivity derived from chlamydiae as compared with the initial homogenate. Several lines of evidence showed that the structures isolated on dextran gradients were chlamydial phagosomes. These structures and free chlamydiae banded at different positions on discontinuous sucrose gradients. The difference was destroyed by the nonionic detergent Nonidet P-40, which disrupts plasma membranes but has no effect on C. psittaci. Material labeled on the surface of the L-cell plasma membrane cosedimented with the phagosome fractions. Electron microscopy of these fractions revealed structures having the appearance of a chlamydial elementary body surrounded by a unit membrane. Sodium dodecyl sulfate-polyacrylamide gels of the phagosome membranes displayed 10 major protein bands, less than the total number of surface-labeled proteins in the L-cell plasma membrane. Seven of the proteins of phagosome membranes had electrophoretic mobilities corresponding to those of proteins exposed on the surface of L cells. Two of them were cleaved by both trypsin and chymotrypsin, enzymes that decrease the susceptibility of L cells to infection with C. psittaci. These proteins may therefore be involved in the attachment and ingestion of C. psittaci by L cells.

δ-Crystallin is a chick lens fiber cell membrane extrinsic protein

In an attempt to resolve the present controversy concerning the reported presence of δ-crystallin in preparations of chick lens plasma membranes, a study was conducted on the extractability of chick lens fiber cell plasma membranes in the presence or absence of calcium ions. One lens from each animal was homogenized in calcium-containing buffer and the other in (calcium) chelating buffer. Each resulting water insoluble crude cell membrane fraction was evenly divided and membranes isolated from each portion by treatment with urea in either calcium-containing buffer or chelating buffer. The isolated membranes were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Gels of membranes isolated in the presence of calcium showed very prominent δ-crystallin bands while gels of membranes isolated under chelating conditions showed a marked depletion of δ-crystallin. The treatment sequence employed ruled out the possibility that the observed binding of δ-crystallin to the membranes was a result of a spurious association caused by calcium because excess soluble δ-crystallin was removed in discarded chelating buffer washes prior to the isolation of the membranes with urea in chelating buffer. The results of the study showed that δ-crystallin is an extrinsic protein of the chick lens fiber cell plasma membranes, and that its linkage to the membranes is calcium-dependent.