Identification of N-linked Glycoproteins in Silkworm Serum Using Con A Lectin Affinity Chromatography and Mass Spectrometry.

Glycosylation is one of the most common post-translational modifications to occur during protein biosynthesis, but remains poorly understood in insects. In this study, we collected serum proteins from two silkworm developmental stages, namely day 7 of the fifth instar larval stage and day 2 of the pupal stage. Results of SDS-PAGE and periodic acid-Schiff staining revealed that most serum proteins with high abundance were putative glycoproteins. LC-MS/MS identified 149 larval and 303 pupal serum proteins in the Con A lectin-enriched fractions. GO analysis revealed that many serum proteins were involved in the proteolysis and carbohydrate metabolic process. 82 N-linked glycoproteins with at least one glycosylation site were identified. N-Linked glycosylation occurred at the sequon, Asn-X-Ser/Thr, and the proportions of Ser and Thr glycosylation at the hydroxy position were found 39.6% and 60.3%, respectively. The N-glycan structures found in serum glycoproteins were mainly Man2FucGlcNAc2 (67.9%). Since storage protein 1 and transferrin had a relatively high abundance in the serum and could be significantly enriched by Con A lectin, their glycosylation was analyzed in detail. Glycoside hydrases, serine proteases and serpins were found to form three interacting glycoprotein networks using the website STRING. This study provides important clues for the understanding of the function of N-linked glycosylation in metabolism, immunity, and metamorphosis.

Characterization of Human Spermatic Subpopulations by ConA-Binding Sites and Tyrosine Phosphorylation during in vitro Capacitation and Acrosome Reaction.

Spermatozoa capacitation is a time-dependent physiological process essential for acquiring the fertilizing capacity. In this context, reorganization of spermatozoa surface sugars and tyrosine phosphorylation are some of the most important biochemical changes involved in capacitation. However, the relationship between these 2 biomarkers remains poorly defined. By cytofluorescence we simultaneously characterized the head concanavalin A (ConA)-binding sites and the flagellar tyrosine phosphorylation before capacitation, during different capacitation times (1 and 4 h), and after acrosome reaction induction in human spermatozoa. The results showed a strong connection between ConA-label patterns and tyrosine phosphorylation according to the spermatozoa capacitation time and acrosomal status. Specifically, the spermatozoa subpopulation with phosphotyrosine presented proper sugar location (label in acrosomal and postacrosomal region) just after 1 h of capacitation, while cells without phosphotyrosine needed 4 h to do it. Moreover, after induction of spermatozoa acrosome reaction, phosphorylation was significantly correlated (p < 0.05) with the relocation of ConA-binding residues to the equatorial region, regardless of capacitation time. Overall, these observations provide novel insights regarding spermatozoa subpopulations based on essential physiological events like capacitation and acrosome reaction, which could have potential implications in the improvement of spermatozoa selection techniques.

Paper analytical devices for dynamic evaluation of cell surface N-glycan expression via a bimodal biosensor based on multibranched hybridization chain reaction amplification.

A novel colorimetric/fluorescence bimodal lab-on-paper cyto-device was fabricated based on concanavalin A (Con A)-integrating multibranched hybridization chain reaction (mHCR). The product of mHCR was modified PtCu nanochains (colorimetric signal label) and graphene quantum dot (fluorescence signal label) for in situ and dynamically evaluating cell surface N-glycan expression. In this strategy, preliminary detection was carried out through colorimetric method, if needed, then the fluorescence method was applied for a precise determination. Au-Ag-paper devices increased the surface areas and active sites for immobilizing larger amount of aptamers, and then specifically and efficiently captured more cancer cells. Moreover, it could effectively reduce the paper background fluorescence. Due to the specific recognition of Con A with mannose and the effective signal amplification of mHCR, the proposed strategy exhibited excellent high sensitivity for the cytosensing of MCF-7 cells ranging from 100 to 1.0×10(7) and 80-5.0×10(7) cellsmL(-1) with the detection limit of 33 and 26 cellsmL(-1) for colorimetric and fluorescence, respectively. More importantly, this strategy was successfully applied to dynamically monitor cell-surface multi-glycans expression on living cells under external stimuli of inhibitors as well as for N-glycan expression inhibitor screening. These results implied that this biosensor has potential in studying complex native glycan-related biological processes and elucidating the N-glycan-related diseases in biological and physiological processes.

Profiling of concanavalin A-binding glycoproteins in human hepatic stellate cells activated with transforming growth factor-ß1.

Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA) was increased in human hepatic stellate cells (HSCs) following activation by transforming growth factor-ß1 (TGF-ß1); however, little is known about the ConA-binding glycoproteins (CBGs) of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-ß1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin) and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase ß-2 [PLCB2]) were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility.

Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

Isolation and identification of Concanavalin A binding glycoproteins from human seminal plasma: a step towards identification of male infertility marker proteins.

Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma.

Ligand accessibility to receptor binding sites enhanced by movable polyrotaxanes.

Functionalized polyrotaxanes are utilized to investigate the relation to multivalent interactions between the mannose moiety and Con A immobilized surfaces. According to the results of SPR spectroscopy, the mannose-conjugated polyrotaxanes show a higher response than any other mannose conjugate on both surfaces of high- and low-density Con A. Moreover, the results of the FRET analysis suggest that the mobility of α-cyclodextrins in the polyrotaxane more efficiently contributes to their binding interactions in a multivalent manner. This well-defined polyrotaxane system provides control over ligand density, ligand mobility, and gives an efficient response to the biological interaction receptor, which has not been easy to achieve in covalently bound polymeric systems.

S-layer, surface-accessible, and concanavalin A binding proteins of Methanosarcina acetivorans and Methanosarcina mazei.

The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often posttranslationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over 100 proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus, minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed nonspecifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically localized. This approach provides an alternative strategy to study surface proteins in the archaea.

Immunosuppresive role of principal toxin (crotoxin) of Crotalus durissus terrificus venom.

The composition of the crotalic venom and the immunochemistry and/or pathophysiological characterization and main components were well studied. However, few studies have been carried out to investigate the effect of toxins of this venom on the development of the immune response. The objective of this work was to find out if venom or crotoxin of Crotalus durissus terrificus was able to modulate the immune response through its ability to change the mediators involved in the immune response by an unrelated antigen. We observed in the murine model, that venom as well as crotoxin have inhibitory effect on splenic cells proliferation induced by Con-A. Moreover, CB did not inhibit the proliferative response, suggesting that the integrity of crotoxin complex is necessary for the development of this phenomenon. Moreover, we showed that the effect on cellular proliferation was unrelated to cytotoxicity activity. We also observed that venom or crotoxin inhibited cytokine release induced in HSA immunised mice, mainly IL-2, IL-4 and IL-10, however, crotoxin did not inhibit the release of IFN-gamma. The involvement of T or B cells in the suppressive effect of venom was evaluated through the transference of purified splenic cells from venom-mice to normal mice that also produced low IgG1 anti-HSA levels, indicating the participation of these cells in this process. Mechanism of action of the crotalic venom on development of immune response to an unrelated antigen is much more complex, therefore it must not only involve the interaction of distinct cellular populations, but activation or inhibition of signalling proteins, need to be further investigated.

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis.

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.

Tail chimeras of Dictyostelium myosin II support cytokinesis and other myosin II activities but not full development.

Dictyostelium lacking myosin II cannot grow in suspension culture, develop beyond the mound stage or cap concanavalin A receptors and chemotaxis is impaired. Recently, we showed that the actin-activated MgATPase activity of myosin chimeras in which the tail domain of Dictyostelium myosin II heavy chain is replaced by the tail domain of either Acanthamoeba or chicken smooth muscle myosin II is unregulated and about 20 times higher than wild-type myosin. The Acanthamoeba chimera forms short bipolar filaments similar to, but shorter than, filaments of Dictyostelium myosin and the smooth muscle chimera forms much larger side-polar filaments. We now find that the Acanthamoeba chimera expressed in myosin null cells localizes to the periphery of vegetative amoeba similarly to wild-type myosin but the smooth muscle chimera is heavily concentrated in a single cortical patch. Despite their different tail sequences and filament structures and different localization of the smooth muscle chimera in interphase cells, both chimeras support growth in suspension culture and concanavalin A capping and colocalize with the ConA cap but the Acanthamoeba chimera subsequently disperses more slowly than wild-type myosin and the smooth muscle chimera apparently not at all. Both chimeras also partially rescue chemotaxis. However, neither supports full development. Thus, neither regulation of myosin activity, nor regulation of myosin polymerization nor bipolar filaments is required for many functions of Dictyostelium myosin II and there may be no specific sequence required for localization of myosin to the cleavage furrow.

Control of multivalent interactions by binding epitope density.

Receptor clustering by multivalent ligands can activate signaling pathways. In principle, multivalent ligand features can control clustering and the downstream signals that result, but the influence of ligand structure on these processes is incompletely understood. Using a series of synthetic polymers that vary systematically, we studied the influence of multivalent ligand binding epitope density on the clustering of a model receptor, concanavalin A (Con A). We analyze three aspects of receptor clustering: the stoichiometry of the complex, rate of cluster formation, and receptor proximity. Our experiments reveal that the density of binding sites on a multivalent ligand strongly influences each of these parameters. In general, high binding epitope density results in greater numbers of receptors bound per polymer, faster rates of clustering, and reduced inter-receptor distances. Ligands with low binding epitope density, however, are the most efficient on a binding epitope basis. Our results provide insight into the design of ligands for controlling receptor-receptor interactions and can be used to illuminate mechanisms by which natural multivalent displays function.

Binding affinity properties of dendritic glycosides based on a beta-cyclodextrin core toward guest molecules and concanavalin A.

The inclusion behavior and concanavalin A binding properties of hepta-antennated and newly synthesized tetradeca-antennated C-6-branched mannopyranosyl and glucopyrannosyl cyclomaltoheptaose (beta-cyclodextrin) derivatives have been evaluated by isothermal titration microcalorimetry and enzyme-linked lectin assay (ELLA), respectively. The synthesis of three first-order dendrimers based on a beta-cyclodextrin core containing 14 1-thio-beta-D-glucose, 1-thio-beta-mannose, and 1-thio-beta-rhamnose residues was performed following a convergent approach and involving (1) preparation of a thiolated bis-branched glycoside building block and (2) attachment of the building block onto heptakis(6-deoxy-6-iodo)-beta-cyclodextrin. Calorimetric titrations performed at 25 degrees C in buffered aqueous solution (pH 7.4) gave the affinity constants and the thermodynamic parameters for the inclusion complex formation of these beta-cyclodextrin derivatives with guests sodium 8-anilino-1-naphthalensulfonate (ANS) and 2-naphthalenesulfonate. The host capability of the persubstituted beta-cyclodextrins decreased with respect to the native beta-CD when sodium 2-naphthalenesulfonate was used as a guest and improved when ANS was used as a guest molecule. Heptavalent mannoclusters based on beta-CD cores enhance the lectin binding affinity due to the cluster effect; however, the increase of the valency from 7 to 14 ligands did not contribute to the improvement of the concanavalin A binding affinity. In addition, the synthesized hyperbranched mannoCDs lost completely the capability as a host molecules.

The distribution of MUC1, an apical membrane glycoprotein, in mammary epithelial cells at the resolution of the electron microscope: implications for the mechanism of milk secretion.

The distribution of the glycoprotein, mucin 1 (MUC1), was determined in lactating guinea-pig mammary tissue at the resolution of the electron microscope. MUC1 was detected on the apical plasma membrane of secretory epithelial cells, the surface of secreted milk-fat globules, the limiting membranes of secretory vesicles containing casein micelles and in small vesicles and tubules in the apical cytoplasm. Some of the small MUC1-containing vesicles were associated with the surfaces of secretory vesicles and fat droplets in the cytoplasm. MUC1 was detected in much lower amounts on basal and lateral plasma membranes. By quantitative immunocytochemistry, the ratio of MUC1 on apical membranes and milk-fat globules to that on secretory vesicle membranes was estimated to be 9.2:1 (density of colloidal gold particles/microm membrane length). The ratio of MUC1 on apical membranes compared with basal/lateral membranes was approximately 99:1. The data are consistent with a mechanism for milk-fat secretion in which lipid globules acquire an envelope of membrane from the apical surface and possibly from small vesicles containing MUC1 in the cytoplasm. During established lactation, secretory vesicle membrane does not appear to contribute substantially to the milk-fat globule membrane, or to give rise in toto to the apical plasma membrane.

Presence of aberrant tumor-reactive immunoglobulins in the circulation of patients with ovarian cancer.

OBJECTIVES: Cancer patients generally exhibit circulating tumor-reactive immunoglobulins; however, these antibodies fail to eradicate tumors or prevent their progression. This study identifies and characterizes an aberrant tumor-reactive IgG population present in women with ovarian cancer. METHODS: In this pilot study, IgG was isolated from the sera of women with advanced-stage ovarian cancer (stages III and IV, n = 62) and age-matched female volunteers (n = 50) by affinity chromatography. These IgGs were characterized on the basis on their aberrant binding to concanavalin A affinity columns. Subsequently, the concanavalin A-binding moiety was localized following IgG fragmentation, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and characterized by oligosaccharide profiling. RESULTS: The level of concanavalin A-binding IgG in our control population was 8.9 +/- 2.9%, whereas in ovarian cancer patients, the level of concanavalin A-binding IgG was 38.8 +/- 7.4%. In the patients with ovarian cancer, 87.5 +/- 5.7% of the tumor-reactive IgG was demonstrated to be concanavalin A-binding. Based on oligosaccharide profiling of the fragmented concanavalin A-binding IgG, the aberrant lectin binding appeared to be the consequence of altered glycosylation of one of the two Fc chains. CONCLUSIONS: While our previous studies have identified the presence of circulating IgG reactive with specific tumor-associated antigens and its association with poor prognosis, this report demonstrated the presence of an aberrantly glycosylated IgG population in cancer patients. This altered IgG appeared to be the primary class of tumor-reactive antibodies in these women.

Increased activity in the biliary Con A-binding fraction accounts for the difference in crystallization behavior in bile from Chilean gallstone patients compared with Dutch gallstone patients.

Chile has one of the highest prevalences of cholesterol gallstone disease in the world. Recent data indicate that this is partly caused by genetic (Indian) factors. However, the causal factors inducing increased gallstone formation have not been elucidated. The aim of this study was to compare biliary composition and cholesterol crystallization in bile from patients of high and moderate risk areas (Chile and The Netherlands) for gallstone disease. Bile was sampled at cholecystectomy from 30 Chilean and 26 Dutch gallstone patients. The Con A-binding fraction (CABF) was extracted from fresh native bile samples by incubation with Con A-sepharose. Reconstitution of the CABF to the Con A-extracted native bile induced almost full recovery of crystallization confirming the validity of this technique. There was no difference between the two groups regarding sex and age. Chilean bile nucleated significantly faster (3.5 +/- 0.6 vs. 7.9 +/- 1.5 days) despite the fact that Dutch bile had a significantly higher cholesterol saturation index (CSI) (1.6 vs. 1.2, P = .01). The total lipid content was not different. Chilean bile contained more total protein (5 vs. 2.9 mg/mL, P = .008). IgG, IgM, Haptoglobin and alpha-1-acid glycoprotein were not different between the two groups. IgA, though, was significantly higher in the Chilean samples (0.44 vs. 0.19 mg/mL, P < .001). Extraction of CABF increased crystal observation time (COT) and decreased crystal growth in both groups. However, the effects were much more pronounced in the Chilean samples. Compared with Dutch bile, Chilean bile crystallizes much faster despite a lower CSI. Chilean bile contains an increased content of Con A-binding nucleation promoting activity.

Change of oligosaccharides of rat brain microsomes depending on dietary fatty acids and learning task.

We have analyzed oligosaccharide chains in brain microsomes of rats fed an n-3 polyunsaturated fatty acid-deficient (safflower oil group; S group) or -rich (perilla oil group; P group) diet before and after brightness-discrimination learning tasks. The amount of concanavalin A-binding sites (mainly mannoside) of the brain microsomes was found to be significantly less in the S group than the P group before the learning task. Detailed analysis of glycoprotein glycans demonstrated that high mannose type oligosaccharides were dominant in brain microsomes before the learning task in both dietary groups, whereas multiantennary complex-type oligosaccharides became dominant after the learning task and especially a tetra-antennary glycan, that had a core structure of the glycan of neural cell adhesion molecule, was more increased in the S-group than the P group. When polysialylated glycans were analyzed on serotonin-conjugated HPLC column, the glycans in the S-group microsomes before the learning task contained larger amount of higher affinity-polysialylated glycans to serotonin column than those in the P-group, and also contained larger amount of phosphoglycans that showed also high affinity to serotonin column than the P-group. Removal of mannoside from microsomes by alpha-mannosidase-treatment changed the membrane surface physical property, especially permittivity, as revealed by analysis of the interaction with 1-anilinonaphthalene-8-sulfonate. These results suggest that high mannose content and several multiantennary glycans including polysialylated and phospho-glycans were changed by dietary n-3 fatty acid deficiency and learning task in rat brain microsomal glycoproteins and that these changes may affect membrane functions through changes of membrane surface physical properties and reactivity against serotonin.

Immunosuppressive acidic protein (IAP) level used to monitor adjuvant arthritis in rats.

To determine if immunosuppressive acidic protein (IAP) responds to the degree of inflammation in adjuvant arthritis in rats (AA), we measured the serum IAP level of the rats by using the single immunodiffusion method, and the level of concanavalin A (Con A) binding protein by using nepherometry. Pharmacologic treatment with 0.3mg/kg of methotrexate (MTX) was done from day 5 to 14 postimmunization in rats with AA. The measured serum IAP level on day 21 indicated the most severe inflamed phase. IAP levels reflected the degree of primary and secondary inflammatory phases in AA, whereas ConA binding protein (CBP) levels did not. Our results suggest that IAP can be used to monitor the symptoms of inflammation and the efficacy of anti-inflammatory drugs.

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells.

The effects of microgravity on Jurkat cells--a T-lymphoid cell line--was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a or 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.

Age-dependence of the lateral diffusion coefficient of Con-A receptor protein in the skeletal muscle membrane of C57BL/6J mice.

The lateral diffusion coefficient (Dp) of the Con-A receptor protein was measured in the sarcolemma of the quadriceps femoris (QF) muscle of male and female C57BL/6JNia mice in four age groups between 2 and 26 months. Freshly prepared, ex vivo taken muscle strips were stained with Con-A-FL conjugate for 10 min, and fluorescence recovery after photobleaching (FRAP) measurements were carried out on 20-30 cells per animal, at 37 degrees C. Using this technique, Dp, and the fractional recovery (mobile fraction = FR%) of these proteins can be measured. In the youngest male and female age groups, Dp values of 5.72E-10 and 5.43E-10 cm2/s, and FR% values of 43.3 and 36.3%, were found, respectively. Dp displayed a characteristic, significant, negative, linear correlation with age in both sexes. The slope of the linear regression line calculated per month of age was 1.06E-11 and 0.96E-11 cm2/s for males and females, respectively; both of them differ from zero highly significantly. FR% values tended to increase slightly with age, yet the estimated average Dp = D(FR), calculated for the total Con-A receptor pool, maintained its significant, negative, linear age-correlation. The physiological significance of these changes needs to be clarified in the future.