Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Of Proteins Research Articles

Sperm protein markers for Holstein bull fertility at National Artificial Insemination Centers in Indonesia.

Background and Aim:Holstein cows and heifers are widely bred in Indonesia by artificial insemination (AI) to increase population and milk production. Sperm fertility is modulated by genetic factors, but the analysis of sperm quality is still based on macro- and microscopic characteristics. This study aimed to analyze both sperm quality and proteins of Holstein bulls at different fertility levels.Materials and Methods:The frozen semen samples were collected from the Indonesia National AI Center. They were classified based on the reproductive efficiency data and were grouped into high fertile (HF) and low fertile (LF). Sperm qualities were evaluated by microscopic evaluation. The Holstein sperm proteins were extracted using phenylmethanesulfonyl fluoride as a protease inhibitor and the benzidine detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to analyze the molecular weights (MWs) of the sperm proteins. The data obtained were analyzed by a t-test using the one-factor bull fertility level, and Spearman’s correlation analysis was used to identify the correlation between the sperm microscopic evaluation parameters and protein bands.Results:The sperm motility post-freeze thawing was not significantly different between the HF and LF (p>0.05). The HF level had a higher percentage of viability, intact plasma membrane integrity, and intact acrosomes than the LF (p<0.05). Five protein bands were found in the SDS-PAGE of sperm proteins of Holstein bulls with different concentrations. Sperm proteins with MWs of 17.51 kDa, 14.87 kDa, 33.71 kDa, and 41.97 kDa were abundant in the Holstein bulls with an HF level, while 55 kDa proteins were abundant in the LF level of Holstein bulls. The sperm of Holstein bulls in the HF level contained proteins of about 33.71 kDa that were not detected in the LF.Conclusion:The sperm protein with a molecular weight of 33.71 kDa was predicted to be a specific protein biomarker that influences bull fertility. Sperm fertilization abilities were also determined by the sperm proteins, the morphology of sperm acrosomes, and the quality of plasma membranes. This method can be used to select bulls with high fertility to increase the population of Holstein bulls.

Electrophoretic profile of seminal proteins and their correlation with in vitro sperm characters in Black Bengal buck semen.

Aim:This study aimed to study the electrophoretic properties of seminal plasma and sperm proteins of Black Bengal buck semen and their correlation with in vitro sperm characters and freezability.Materials and Methods:Semen ejaculates from nine Black Bengal bucks were collected by artificial vagina (n=20/buck). Ejaculates were evaluated for in vitro sperm characters and electrophoretic profile of seminal protein. In vitro sperm characters were evaluated immediately after collection, after completion of equilibration period, and after freeze-thawing. For seminal protein studies, seminal plasma proteins were precipitated by ice-cold ethanol method, and sperm proteins were extracted by Triton X detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess the molecular weight of seminal proteins. Correlation between in vitro sperm characters and protein bands was determined by Pearson’s correlation coefficient, and two-way ANOVA was applied to find the individual buck differences.Results:Significant difference (p<0.01) among the bucks was noticed in the in vitro sperm characters evaluated at all the three stages of semen evaluation such as immediately after collection, after completion of equilibration period, and post-freeze thawing. Progressive loss of sperm motility, membrane integrity, and other in vitro sperm characters were noticed during cryopreservation. A total of ten protein bands in the molecular weight ranging from 17 to 180 kDa were found in the SDS-PAGE of seminal plasma proteins, while nine bands of 17-134 kDa were observed in sperm proteins. Seminal plasma proteins of molecular weight 75, 62-49, 20, and 17 kDa and sperm proteins of 75, 20, and 17 kDa were present in all the nine bucks (100%) screened, and variation among the bucks was noticed for the presence of other proteins. Seminal plasma protein of 180-134 kDa showed a negative correlation with individual motility (−0.716) and functional membrane integrity of sperm cells (−0.724) in post-freeze–thaw analysis and 48 kDa protein had a positive correlation with individual motility (0.649) and functional membrane integrity of sperm cells (0.664) in post-thaw analysis. Sperm proteins of 63 kDa had a negative correlation (−0.616) with sperm concentration in neat semen.Conclusion:Variation among the bucks was noticed in the in vitro sperm characters and semen freezability. Correlation between seminal proteins and in vitro sperm characters and semen freezability had been found which might be useful as a tool to select breeding bucks.

Determination of Protein Molecular Weights on SDS-PAGE.

An apparent molecular weight (MW) of a protein can be determined from the migration distance of a protein complexed with a strong cationic detergent sodium dodecyl sulfate (SDS) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This method was established around 1969 and has been utilized substantially even today because of its simplicity. During the following half a century, although it has been reported that many proteins show some deviation in MW when determined on SDS-PAGE especially when their peptide chains are posttranslationally modified, this versatile method is still being used very often in current biochemical works. In this protocol, a simple method to estimate MW by running SDS-PAGE of standard proteins is explained by an example in which proteins extracted from mouse retina were analyzed by two-dimensional isoelectric focusing (2-D IEF) SDS-PAGE followed by protein identification by peptide mass fingerprinting.

Effect of Corilagin on Membrane Permeability of Escherichia coli, Staphylococcus aureus and Candida albicans

Corilagin is a member of polyphenolic tannins. Its antimicrobial activity and action mechanism against Escherichia coli, Staphylococcus aureus and Candida albicans were investigated through membrane permeability. Crystal violet staining determination, outer membrane (OM) and inner membrane (IM) permeability, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and atomic force microscopy (AFM) were used as methods for our investigation. The minimum inhibitory concentrations were 62.5, 31.25 and 62.5 µg/mL for E. coli, S. aureus and C. albicans, respectively. Crystal violet results and SDS-PAGE of supernatant proteins showed that corilagin dose-dependently affected membrane permeability of E. coli and C. albicans but not of S. aureus. OM and IM permeability assays revealed comparable results for E. coli. By using AFM, we demonstrated extensive cell surface alterations of corilagin-treated E. coli and C. albicans. SDS-PAGE of precipitated proteins revealed possible targets of corilagin, i.e. Fib, Sae R, Sar S in S. aureus and Tye 7p in C. albicans. In conclusion, corilagin inhibited the growth of E. coli and C. albicans by disrupting their membrane permeability and that of S. aureus by acting on Fib, Sae R and Sar S but not on membrane integrity.

Tuber quality assessment of orange-fleshed sweet potato (Ipomoea batatas) cultivars and their genetic relatedness as revealed by SDS-PAGE of tuber proteins

Orange-fleshed sweet potato (OFSP) is an important source of β-carotene and can make a major contribution in alleviating vitamin A malnutrition. Replacement of white-fleshed varieties with new b-carotene-rich OFSP cultivars that meet local preference would benefit the children who are at risk for vitamin A deficiency related problems. Twelve OFSP cultivars of diverse origin were analyzed for biochemical and antioxidant composition, viz dry matter, crude protein, crude fat crude fibre, carbohydrate, starch, amylose, reducing sugars, total phenols, ascorbic acid and b-carotene contents of their tubers. Many of the investigated cultivars have high levels of dry matter, crude protein, starch, β-carotene, phenols and ascorbic acid. Cultivars with high dry matter and b-carotene can be utilized to develop cultivars for commercial exploitation. This paper also highlights the genetic relatedness among the OFSP cultivars, assessed by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of tuber proteins. High genetic similarities were observed among the cultivars originated in India.

A simple and rapid method for the differentiation and identification of thermophilic bacteria

In total, 170 strains of thermophilic bacteria were isolated from deep-sea hydrothermal fields in the Pacific Ocean and a hot spring in Xiamen of China. To facilitate the identification of thermophilic strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins of these strains was first performed. The results showed that there exist four different protein patterns, indicating that the 170 strains might belong to four species or genera. The RAPD (random amplified polymorphic DNA) profiles of nine representative strains were consistent with those of SDS-PAGE. To further identify the species of the nine strains, their 16S rDNA sequences were analyzed. The results showed that the nine strains fell into four species of three genera, which was the same as revealed by SDS-PAGE. Therefore, SDS-PAGE of whole-cell proteins could be used as a rapid and simple method for the discrimination of thermophilic bacteria as the first step of species identification.

Reclassification of Paenibacillus (formerly Bacillus) pulvifaciens (Nakamura 1984) Ash et al. 1994, a Later Subjective Synonym of Paenibacillus (formerly Bacillus) larvae (White 1906) Ash et al. 1994, as a Subspecies of P. larvae, with Emended Descriptions of P. larvae as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens

A polyphasic taxonomic study of four strains of Paenibacillus larvae and four strains of Paenibacillus pulvifaciens (including duplicates of both type strains) supported the reclassification of both former Bacillus species into one species, P. larvae. Our conclusions were based on morphological and Analytab Products (API) tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins, gas chromatography of methylated fatty acids, pyrolysis mass spectrometry, DNA-DNA binding, and the following genomic fingerprinting methods: amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and AFLP analysis. The last method is a novel high-resolution DNA fingerprinting technique based on the selective amplification of restriction fragments. Despite more than 90% DNA relatedness between the strains studied, SDS-PAGE of whole-cell proteins, biochemical tests, and DNA fingerprinting (AFLP) distinguished between the P. larvae and P. pulvifaciens strains at the subspecies level. Taking this evidence along with differences in pathogenicity, we propose to reclassify the honeybee pathogens P. larvae and P. pulvifaciens as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens. An emended description of the species and descriptions of the subspecies are given. The type strains are P. larvae subsp. larvae ATCC 9545 (LMG 9820) and P. larvae subsp. pulvifaciens NRRL B-3685 (LMG 6911 and ATCC 13537).

Characterization of Legionella species by numerical analysis of whole-cell protein electrophoresis.

The results of a computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 291 isolates and 74 reference strains belonging to all known species of the genus Legionella revealed that the majority of the species of this genus can be adequately identified by this method. The type strain of Legionella bozemanii did not cluster with the other strains of this species, and the only strain of Legionella geestiana available clustered with the strains of Legionella feeleii. When we performed a numerical analysis by omitting certain portions of the pattern containing dense bands, all of the species could be distinguished. Our results also show that the type strains of Legionella nautarum and Legionella londiniensis deposited in the National Collection of Type Cultures do not correspond to the type strains deposited in the American Type Culture Collection. We used the results of a fatty acid and ubiquinone composition analysis to complement the SDS-PAGE results for several strains whose identities as determined by indirect immunofluorescence were doubtful. Computer-assisted SDS-PAGE of whole-cell proteins can be used in the classification of Legionella species and to identify and screen large numbers of isolates for further, in-depth taxonomic studies of smaller numbers of strains.

Protein Alterations in Age-related Cataract Associated with a Persistent Hyaloid Vascular System in Senescence-accelerated Mouse (SAM)

The occurrence of age-related cataract associated with a persistent hyaloid vascular system is the most prominent feature in SAMP9, an inbred strain of Senescence-accelerated Mouse. To examine the cataractogenesis, we analysed protein changes in the process of cataract formation in the lens. The cataractous lenses showed a striking decrease in water-soluble protein content, in contrast to increases in the amount of water insoluble protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots of water-soluble protein in the cataractous lenses showed additional high molecular weight β-crystallin proteins of about 43 kDa. concomitant with decreased amounts of 29-kDa and 31-kDa β-crystallins and 21-kDa γ-crystallin, as compared with findings in normal lenses. Although there was no apparent difference between the patterns of SDS-PAGE of urea-soluble and urea-insoluble proteins isolated from cataractous and normal lenses, slightly increased reactivity of bands around 43 kDa against anti-β-crystallin antibody was observed in cataractous lenses. The calcium content was elevated and activity of transglutaminase was increased in the cataractous lenses. While the molecular weight of β-crystallin polymers cross-linked in vitro by exogenous transglutaminase was not completely compatible with those of high molecular weight β-crystallins observed in the cataractous lenses, these findings do suggest the contribution of this enzyme to production of high molecular weight β-crystallins and to insolubilization of these proteins in the cataractous lenses in SAMP9.

Phenotypic and Genotypic Characterization of Bradyrhizobia Nodulating the Leguminous Tree Acacia albida

Rhizobial isolates that were obtained from both surface and deep soil samples in the Sahelian and Sudano-Guinean areas of Senegal (West Africa) under Acacia albida trees were compared with representative strains of known rhizobial species and genera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was used to determine the taxonomic positions of these organisms and the relationships between isolates obtained from the surface and isolates obtained from deep soil. Most of the isolates belonged to eight electrophoretic clusters containing representative strains of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and Bradyrhizobium sp. Isolates were also characterized by the Biolog system, and the results were compared with the results obtained by SDS-PAGE of total proteins; the level of correlation was very low. DNA-rRNA hybridizations with 16S or 23S rRNA from Bradyrhizobium japonicum LMG 6138T (T = type strain) confirmed that most of the protein electrophoretic clusters belong in the Bradyrhizobium-Rhodopseudomonas rRNA complex. Sequencing of 16S rRNA genes showed that some of the A. albida-nodulating isolates belong to a separate lineage together with representatives of other protein electrophoretic clusters. Other isolates that belong to the same electrophoretic cluster as the type strain of Bradyrhizobium japonicum are considered members of the lineage represented by this type strain. The first lineage is as far removed from Bradyrhizobium japonicum as it is from the genus Afipia, Blastobacter denitrificans, and the genus Rhodopseudomonas. The possible relationship among electrophoretic group, geographic origin, and depth of isolation at a particular site is discussed.

Characterization of Rhizobia Isolated from Different Divergence Groups of Tropical Leguminosae by Comparative Polyacrylamide Gel Electrophoresis of their Total Proteins

Summary In an attempt to determine the taxonomic position of and the relationships between some 800 strains of bradyrhizobia and rhizobia isolated from nodules of tropical leguminous plants in the Amazon region and Atlantic forests of Brazil, 171 strains (of which 120 were slow or very slow growers) were selected for study by sodium dodecyl sulphate polyacrylamide gel electrophoresis of proteins (SDS-PAGE). The strains were chosen to represent culturally different isolates from different divergence groups of Leguminosae. Appropriate type and reference strains and also seven tropical strains isolated from Phaseolus vulgaris were included for comparison. At a mean correlation coefficient (r) of 0.86, 23 protein electrophoretic clusters were obtained. The majority of the isolates grouped in a large cluster that contained the type strain of Bradyrhizobium japonicum. This cluster could be divided in 5 subclusters that correlated only to some degree with previously determined DNA homology groups. A few of the isolates could be equated with the species Rhizobium fredii (syn. Sinorhizobium fredii), R. galegae and R. loti but many could not be allocated to currently described taxa. No correlation was noted between clusters obtained and the divergence groups of Leguminosae from which the strains were isolated.

Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken

The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell-cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P-40-solubilized retina membranes had two components as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 45.5-kDa doublet and a 69-kDa polypeptide. Immunoreactive bands of 46-50 kDa were recognized following SDS-PAGE of detergent-solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N-glycosidase F (EC 3.2.2.18) converted the 45.5-50-kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N-Glycosidase F treatment also converted the 69-kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32-kDa species. Treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5-kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O-glycosidase (endo-alpha-N-acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for alpha 2,6-linked sialic acid) was confined to the higher molecular mass species of the 45.5-kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue-specific posttranslational modifications, particularly differences in sialylation of antigen-bearing polypeptides.

Characterisation of hospital isolates of Moraxella (Branhamella) catarrhalis by SDS-PAGE of whole-cell proteins, immunoblotting and restriction-endonuclease analysis.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins (WCP), immunoblot analysis and DNA restriction-endonuclease analysis (REA) were applied as potential typing methods to 31 clinically significant strains of Moraxella (Branhamella) catarrhalis, five of which came from a suspected outbreak of nosocomial infection in a respiratory-diseases ward. Twelve of 31 isolates were placed in four groups, each of which contained strains indistinguishable by the three typing techniques used. Each of a further two groups contained two strains, and they were similar by at least one technique; the remaining 15 strains were unique by all three methods. Four of five strains from the suspected outbreak were indistinguishable by SDS-PAGE of WCP, immunoblotting and REA. Results show that SDS-PAGE of WCP, immunoblotting and REA are suitable techniques for characterising M. catarrhalis and that there is a considerable degree of strain heterogeneity. Nosocomial infection with M. catarrhalis may be relatively common and further epidemiological studies with a combination of typing techniques are indicated.

Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells

Phenylhydrazine-mediated protein damage in human red cells has been assessed using HPLC, one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis of major membrane proteins. The association of the Ca 2+-activated neutral protease, calpain, with membrane proteins following hydrazine insult was also examined using immunoblot analysis. HPLC amino acid analysis of red cell suspensions was employed to quantify proteolysis. Phenylhydrazine (4 m m) increased the rate of leucine, lysine, and histidine release by ∼12-, 7-, and 5-fold, respectively. N-acetylcysteine (20 m m), dithiothreitol (50 m m), and dimethylthiourea (50 m m) decreased the rate of phenylhydrazine-stimulated amino acid release by ∼30–50%; in contrast, the free radical scavengers and antioxidants dimethylfuran (50 m m) and dimethyl sulfoxide (50 m m) were without significant effect. The calcium chelator, EGTA (10 m m), inhibited phenylhydrazine-stimulated proteolysis by ∼30%. Phenylhydrazine (4 m m) caused attenuation of the major membrane protein bands present in the SDS-PAGE pattern and extensive smearing of a band in the region of ∼28 kDa. Free radical scavengers and antioxidants failed to ameliorate significantly membrane protein damage in phenylhydrazine-treated cells as judged by SDS-PAGE. Immunoblot analysis of spectrin confirmed these results. Two-dimensional SDS-PAGE of membrane proteins following phenylhydrazine treatment, however, revealed the appearance of new protein spots and a loss of existing protein spots as compared to control. Western blot analysis of membrane-associated calpain (79 kDa (proenzyme), 77- and 75-kDa forms) was also performed. Phenylhydrazine-treated red blood cells exhibited concentration- and time-dependent changes in the level of membrane-associated procalpain relative to control. The inhibitors N-acetylcysteine, dithiothreitol, dimethylthiourea, and dimethyl sulfoxide in the presence of phenylhydrazine appeared to preserve the level of procalpain in association with the membrane proteins, but only N-acetylcysteine and dithiothreitol protected the 77- and 75-kDa forms. In contrast, dimethylfuran in the presence of phenylhydrazine caused a substantial decrease in all three forms of membrane-associated calpain. In phenylhydrazine-treated hemolysate, the level of the 77- and 75-kDa forms of membrane-associated calpain was decreased relative to control. These forms were absent when EGTA (10 m m) was included in the incubation and the level of proenzyme was decreased. These data suggest that calpain is recruited to the membrane following hydrazine insult, undergoes a Ca 2+-dependent conversion to the active forms, and may be involved in the degradation of damaged cytosolic and membrane protein(s).

Characterization and purification of the hyaluronan-receptor on liver endothelial cells

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.

Diversity within Iowa Bradyrhizobium japonicum Serogroup 123

AbstractBradyrhizobium japonicum serogroup 123 dominate soybean [Glycine max (L.) Merr.] nodules in the midwestern USA, but the diversity within this group has been little characterized. This study examined three collections of serogroup 123: nine USDA serogroup 123 strains (representing organisms from several states), 98 isolates collected from several counties and soils across Iowa, and 78 isolates from an intensively sampled 22.8‐m2 area in central Iowa. The organisms were characterized by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of proteins (SDS‐PAGE) and growth response to antibiotics, arginine, and succinate. Of the two methods, SDS‐PAGE was more useful for differentiating organisms. By using SDS‐PAGE, 74 strains were identified from 176 Iowa serogroup 123 isolates. No relation was evident between strain distribution and soil characteristics or geography. Strain USDA 123 was found not to be representative of the Iowa isolates.

Posttranslational Protein Modification by Amino Acid Addition in Regenerating Optic Nerves of Goldfish

Previous experiments have demonstrated that 4S RNA, (tRNA), is transported axonally during the reconnection and maturation of regenerating optic nerves of goldfish. The present experiments were performed to determine if tRNA is transported axonally during elongation of these regenerating nerves and whether, as has been demonstrated in other systems, it participates in posttranslational protein modification (PTPM). [3H]Uridine was injected into both eyes of fish with intact optic nerves and 0, 2, 4, or 8 days after bilateral optic nerve cut. Fish were killed 2 days after injection, and [3H]RNA was isolated from retinae and nerves by phenol extraction and ethanol precipitation. [3H]RNA was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the percentage of [3H]4S RNA remained constant in all retinal and control nerve samples, regenerating nerves showed a twofold increase by 6 days after injury, suggesting that [3H]4S RNA is transported axonally in regenerating nerves as early as 6 days after injury. In other experiments, the 150,000-g supernatant of optic nerves was analyzed for incorporation of 3H-amino acids into proteins. No incorporation of 3H-amino acid was found in the soluble supernatant, but when the supernatant was passed through a Sephacryl S-200 column (removing molecules less than 20,000 daltons), [3H]Arg, [3H]Lys, and [3H]Leu were incorporated into proteins. This posttranslational addition of amino acids was greater (1.4-5 times for Lys and 2-13 times for Leu) in regenerating optic nerves than nonregenerating nerves, and the growing tips of regenerating nerves incorporated 5-15 times more [3H]Lys and [3H]Leu into proteins than did the shafts.(ABSTRACT TRUNCATED AT 250 WORDS)

A variant of erythrocyte membrane skeletal protein band 4.1 associated with hereditary elliptocytosis

A family comprising three patients (a mother and two children) with mild hereditary elliptocytosis was studied. Each patient had prominent elliptocytosis, reduced red cell deformability, and normal erythrocyte thermal sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the erythrocyte membranes in each patient showed decreased levels of band 4.1 (approximately half of the normal value) and the presence of an additional band migrating below protein band 4.2. This additional band was shown to derive from protein 4.1. Comparative partial proteolytic mapping of protein 4.1 and the additional band revealed a number of common peptides. Enzyme-linked immunoelectrotransfer blots of the patients' erythrocyte membranes using a monoclonal antibody to protein 4.1 revealed that, in addition to protein 4.1, two other bands below protein 4.2 were stained; one of these bands migrated in the same position as the additional band detected in the Coomassie Blue-stained gels. Immunoblotting of the patients' whole cells using the antibody to protein 4.1 revealed that this altered band 4.1 occurred as such in the intact red cell. SDS-PAGE of protein 4.1 purified from one patient showed the presence of two lower molecular weight bands below protein 4.1; the lower band migrated in the same position as the additional band found on SDS-PAGE of the patients' erythrocyte membranes. The patient's purified protein 4.1 displayed a decrease of about 40% in the binding activity with crude spectrin extracted from normal controls. Spectrin-spectrin interactions were normal in the three patients. The additional band present in the patients' red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients' erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.

A Variant of Erythrocyte Membrane Skeletal Protein Band 4.1 Associated With Hereditary Elliptocytosis

A family comprising three patients (a mother and two children) with mild hereditary elliptocytosis was studied. Each patient had prominent elliptocytosis, reduced red cell deformability, and normal erythrocyte thermal sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the erythrocyte membranes in each patient showed decreased levels of band 4.1 (approximately half of the normal value) and the presence of an additional band migrating below protein band 4.2. This additional band was shown to derive from protein 4.1. Comparative partial proteolytic mapping of protein 4.1 and the additional band revealed a number of common peptides. Enzyme-linked immunoelectrotransfer blots of the patients' erythrocyte membranes using a monoclonal antibody to protein 4.1 revealed that, in addition to protein 4.1, two other bands below protein 4.2 were stained; one of these bands migrated in the same position as the additional band detected in the Coomassie Blue-stained gels. Immunoblotting of the patients' whole cells using the antibody to protein 4.1 revealed that this altered band 4.1 occurred as such in the intact red cell. SDS-PAGE of protein 4.1 purified from one patient showed the presence of two lower molecular weight bands below protein 4.1; the lower band migrated in the same position as the additional band found on SDS-PAGE of the patients' erythrocyte membranes. The patient's purified protein 4.1 displayed a decrease of about 40% in the binding activity with crude spectrin extracted from normal controls. Spectrin-spectrin interactions were normal in the three patients. The additional band present in the patients' red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients' erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.

Effects of lead on the protein of erythrocyte membranes

In order to study the effects of lead in the circulating peripheral blood on the erythrocyte membrane, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of protein in the erythrocyte membrane was performed in 28 men exposed to lead in a scrap lead refining factory and 18 men working in railway construction as the controls. Furthermore, in order to corroborate the effects in vitro experiments by SDS-PAGE of protein in the mixture of human erythrocyte membranes with added lead were performed. 1) In the lead workers compared with the controls, SDS-PAGE of protein in the erythrocyte membrane showed that relative concentrations of bands 1, 2, 3 and 4.1 were significantly decreased while those of bands 2.3, 6 and 7 were significantly increased. 2) SDS-PAGE of protein in the in vitro experiment showed that the relative concentrations of bands 1, 2, 3, 4.1 and 4.2 were decreased while those of bands 2.3, 4.5, 5, 6 and 7 were increased. Therefore, these results show the same trends as those of the in vivo experiments. 3) For the lead workers, the correlation coefficients between blood lead and bands 2, 2.3 and 3 were r = -0.414 (p less than 0.05), r = 0.545 (p less than 0.01) and r = -0.509 (p less than 0.01), respectively. Also a higher coefficient of correlation (r = -0.777, p less than 0.001) was found between bands 2 and 2.3. 4) As the molecular weight of substances showing bands 2 and 2.3 were 220700 and 133800, respectively, according to PAGE using molecular weight markers, band 2.3 is considered to be due to a cleavage product from that of band 2. From these results, we summarized as follows. Lead combined with erythrocyte membrane decreases the band 3 penetrating lipid bilayer, and that causes the decrease of membrane transportation. On the other hand, the variation of band 2 and 4.1 influences the spectrin-actin network. This is considered to be one of the reasons that MCV is decreased.