Biomedical subjects

S C Braunagel

Publications and source records attributed to S C Braunagel.

At least 19 recordsLinked to original sources

PubMed2003

Determination of the protein composition of the occlusion-derived virus of Autographa californica nucleopolyhedrovirus.

The occlusion derived form of baculovirus is specially adapted for primary infection of the host midgut epithelium. As such, the virion must contain the proteins essential for host range determination and initiation of infection. Because knowledge of virion composition is a prerequisite for functional investigation, this study used a combination of techniques to identify the proteins present within or associated with the occlusion-derived virus (ODV) virion. Thirty-one proteins, including proteins known to be essential for viral DNA replication, were identified with confidence. An additional 13 proteins were identified by using one of the three techniques. A comparison of gene conservation among the ODV proteins encoded in the 16 sequenced baculoviridae genomes is presented. With knowledge of the composition of ODV, it is now possible to target proteins and study their role(s) during primary infection.

Amino Acid Sequence↗

PubMed2001

Effects of deletion and overexpression of the Autographa californica nuclear polyhedrosis virus FP25K gene on synthesis of two occlusion-derived virus envelope proteins and their transport into virus-induced intranuclear membranes.

Partial deletions within Autographa californica open reading frame 61 (FP25K) alter the expression and accumulation profile of several viral proteins and the transport of occlusion-derived virus (ODV)-E66 to intranuclear membranes during infection (S. C. Braunagel et al., J. Virol. 73:8559-8570, 1999). Here we show the effects of a full deletion and overexpression of FP25K on the transport and expression of two ODV envelope proteins, ODV-E66 (E66) and ODV-E25 (E25). Deletion and overexpression of FP25K substantially altered the levels of expression of E66 during infection. Compared with cells infected with wild-type (wt) virus, the levels of E66 were reduced fivefold in cells infected with a viral mutant lacking FP25K (DeltaFP25K) and were slightly increased in cells infected with a viral mutant overexpressing FP25K (FP25K(polh)). In contrast, no significant changes were observed in the levels of E25 among wt-, DeltaFP25K-, and FP25K(polh)-infected cells. The changes observed in the levels of E66 among the different viral mutants were not accompanied by changes in either the time of synthesis, membrane association, protein turnover, or steady-state transcript abundance. Deletion of FP25K also substantially altered the transport and localization of E66 during infection. In cells infected with the DeltaFP25K mutant virus, E66 accumulated in localized regions at the nuclear periphery and the outer nuclear membrane and did not traffic to intranuclear membranes. In contrast, in cells infected with the FP25K(polh) mutant virus E66 trafficked to intranuclear membranes. For comparison, E25 was normally transported to intranuclear membranes in both DeltaFP25K- and FP25K(polh)-infected cells. Altogether these studies suggest that FP25K affects the synthesis of E66 at a posttranscriptional level, probably by altering the translation of E66; additionally, the block in transport of E66 at the nuclear envelope in DeltaFP25K-infected cells suggests that the pathway of E66 trafficking to the inner nuclear membrane and intranuclear microvesicles is specifically regulated and must be influenced by factors that do not control the traffic of E25.

Animals↗

PubMed2001

Identification of BV/ODV-C42, an Autographa californica nucleopolyhedrovirus orf101-encoded structural protein detected in infected-cell complexes with ODV-EC27 and p78/83.

orf101 is a late gene of Autographa californica nucleopolyhedrovirus (AcMNPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (1629K) and ODV-EC27 (orf144). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.

Amino Acid Sequence↗

PubMed1999

Mutations within the Autographa californica nucleopolyhedrovirus FP25K gene decrease the accumulation of ODV-E66 and alter its intranuclear transport.

Previous reports indicate that mutations within the Autographa californica nucleopolyhedrosis virus FP25K gene (open reading frame 61) significantly reduce incorporation of enveloped nucleocapsids into viral occlusions. We report that FP25K is a nucleocapsid protein of both the budded virus (BV) and occluded virus (ODV), and we describe the effects of two FP25K mutations (480-1 [N-terminal truncation] and FP-betagal [C-terminal fusion]) on the expression and cellular localization of ODV-E66 and ODV-E25. Significantly decreased amounts of ODV-E66 are detected in cells infected with 480-1 or FP-betagal viral mutants, even though during FP-betagal infection, steady-state levels of ODV-E66 transcripts remain unchanged. While ODV-E66 is normally detected in intranuclear microvesicles and ODV envelopes by 24 h postinfection (p.i.), ODV-E66 remains cytosolic throughout infection in cells infected with 480-1 virus (up to 96 h p.i.), and its intranuclear localization is not detected until 96 h p.i. in cells infected with the FP-betagal mutant virus. The nuclear localization of ODV-E25 is not affected during infection by the FP-betagal mutant; however, its trafficking is significantly delayed during infection by the 480-1 mutant. Temporal Western blot analyses of cell lysates show that both 480-1 and FP-betagal mutant virus infections result in altered accumulation patterns of several structural proteins, including gp67, BV/ODV-E26, and the major capsid protein p39. In addition to BV/ODV-E26, ODV-E66 and gp67 may interact with FP25K, and ODV-E25 and p39 may also be components of a protein complex containing ODV-E66 and FP25K. Together, these data suggest that FP25K and its associated protein complex(es) may play an important role in the targeting and intracellular transport of viral proteins during infection.

Amino Acid Sequence↗

PubMed1998

The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.

Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.

Amino Acid Sequence↗

PubMed1998

Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase.

Baculovirus infection results in the induction of membrane structures within the nucleoplasm of the host cells. The source of these membranes is unclear; however, using the normal dynamics of cellular membranes and the nuclear envelope as a model, it is possible that the cell cycle might play a role in the regulation of formation of these intranuclear membranes. Therefore, one goal of this study was to investigate the effect of baculovirus infection on the cell cycle of Sf9 host cells. Since few data are available on the cell cycle of insect cells, the first task was to define Sf9 cell cycle kinetics. The cell cycle phase distribution of Sf9 cells grown in suspension culture was determined to be evenly distributed (29% of the cells in G1, 33% in S, and 36% in G2/M phase), with the duration of G1 and S phases both being about 6 h and the combined duration of G2/M phase being about 8 h. When Sf9 cells were infected with AcMNPV (Autographa californica nuclear polyhedrosis virus), approximately 84% of the cells were arrested in G2/M phase by 18-24 h p.i. Concomitant with the viral-induced arrest in G2/M phase, high levels of both cdc2-associated histone H1 kinase activity and cyclin B protein were detected. By 24 h p.i. cyclin B was no longer detected; however, cdc2-associated histone H1 kinase activity remained throughout the infection. These data suggested that early in infection, cyclin B/cdc2 complex may be used to regulate the transition from G2 to M phase, but prolonged arrest may be due to a protein(s) encoded by AcMNPV. DNA hybridization analysis showed that the maximal rate of viral DNA replication occurred before G2/M arrest. We noted that viral DNA replication still occurred late in infection, when the majority of the cells were arrested in G2/M phase. Since cellular DNA replication normally does not occur during G2 or M phase, experiments were designed to determine if viral DNA replication could occur even when host cell DNA replication was arrested. Sf9 cells were arrested and "frozen" at the boundary of G1/S phase using 5-fluoro-2'deoxyuridine (FdUrd) treatment and then infected with AcMNPV In the blocked, infected cells, viral DNA replication was detected; however, cellular DNA remained at steady-state levels. These results suggested that cellular DNA replication was not necessary for viral DNA replication and show that viral DNA replication was not significantly inhibited by FdUrd treatment. It was a surprise to detect viral DNA replication when the host cells were "frozen" at G1/S phase. We wanted to determine if the viral infection was progressing to the stage of progeny virus production. Our data showed that progeny budded virus (BV) and virus-induced intranuclear microvesicles were produced in the frozen, infected cells; however, the intranuclear microvesicles had an unusual structure. They were irregular in shape and thickened compared to those observed in a normal infection. Very few enveloped nucleocapsids were visible in the nucleus of the frozen, infected cells and the occluded-derived virus envelope proteins, ODV-E66 and ODV-EC27, were not detected by Western blot analyses. Since the cells were sustained at the boundary of G1 and S phases for the duration of this experiment, the decreased amount of enveloped ODV in the nucleus could be due to several factors, including decreased levels of proteins expressed from late genes, aberrant microvesicles, or the necessity of G2/M phasing of the infected cell for efficient production and maturation of intranuclear microvesicles. These data indicate that AcMNPV infection results in cell cycle arrest in G2/M phase and this arrest may be due to a viral-encoded protein(s) that has cdc2-associated kinase activity. (ABSTRACT TRUNCATED)

Animals↗

PubMed1998

Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes.

The Autographa californica nuclear polyhedrosis virus da26 gene codes for an envelope protein of both budded virus (BV) and occlusion derived virus (ODV). Western blot and temporal analysis of infected cell extracts detected a protein of 26 kDa by 4 h postinfection (p.i.). The amount of protein increased by 16 h p.i. and remained at high levels throughout infection. By 36 h p.i. several additional immunoreactive proteins were detected which migrated at approximately 18 kDa and remained through 96 h p.i. Western blot analysis of purified virus envelope and nucleocapsid preparations revealed that both the 26- and 18-kDa proteins are structural proteins of the envelope of BV and ODV. Immunoelectron microscopy performed at a time when only the 26-kDa species of the protein was present confirmed that the protein located to ODV envelope. The protein was named BV/ODV-E26 to designate incorporation into viral progeny, envelope location, and apparent molecular weight. Studies designed to follow localization of BV/ODV-E26 demonstrated that early in infection, the protein was incorporated into cytoplasmic vesicles and by 16 h p.i., BV/ODV-E26 was detected in the nucleus associated with virus-induced intranuclear microvesicles and ODV envelope. Coimmunoprecipitation and yeast two-hybrid assays showed that BV/ODV-E26 and FP25K were capable of interacting with each other to form a complex and coimmunoprecipitation assays indicated that cellular actin was a third component of this complex. Together, these data suggest that FP25K and cellular actin may participate in the regulation, or movement through the cell, of baculovirus proteins and/or virus nucleocapsids.

Amino Acid Sequence↗

PubMed1997

N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus.

Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the Autographa californica nuclear polyhedrosis virus (AcMNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to follow protein transport and localization within the nucleus during infection. Autoflourescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized as foci to discrete locations within the nucleus. Immunoelectron microscopy confirmed that these foci predominantly contained intranuclear microvesicles and the reporter fusion proteins were also detected in cytoplasmic membranes near the nucleus, and the outer and inner nuclear membrane. Therefore, these defined hydrophobic domains are sufficient to direct native and fusion proteins to induced membrane microvesicles within a baculovirus-infected cell nucleus and the viral envelope. In addition, these data suggest that movement of these proteins into the nuclear envelope may initiate through cytoplasmic membranes, such as endoplasmic reticulum, and that transport into the nucleus may be mediated through the outer and inner nuclear membrane.

Amino Acid Sequence↗

PubMed1996

Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27.

This paper identifies two structural proteins of the occluded derived viral envelope of Autographa californica nuclear polyhedrosis virus (AcMNPV): ODV-E18 and ODV-E35. In addition, we identify a protein, ODV-EC27, that is incorporated into the capsid of occluded virus, which is not detected in budded virus. The genes for these proteins reside within the IE0 intron. The intron was sequenced, and five open reading frames (ORF) were identified. ORF 3 (genomic ORF 143) codes for the ODV envelope protein, ODV-E18. ORF 4 (genomic ORF 144) codes for ODV-EC27, and Western blot analyses locate this protein to both the ODV capsid and envelope. Transcripts for both ODV-E18 and ODV-EC27 initiate from conserved TAAG motifs, and transcripts are detected from 16 through 72 hr p.i. Antiserum to ODV-E18 recognizes a band of 18 kDa on Western blots of extracts from infected cells and bands of 18 and 35 kDa on Western blots of proteins from purified ODV envelope. N-terminal amino acid sequencing reveals that both ODV-E18 and ODV-E35 contain the same N-terminus. Antiserum to ODV-EC27 recognizes a protein of 27 kDa on Western blots of extracts from infected cells and bands of 27 and 35 kDa on Western blots of proteins from purified ODV. Using immunogold labeling techniques, ODV-E18 and/or ODV-E35 are detected in viral induced intranuclear microvesicles and are not detected in the plasma membrane, cytoplasmic membranes, or the nuclear envelope. Immunogold labeling using antisera to ODV-EC27 detects this protein on both the ODV envelope and capsid.

Amino Acid Sequence↗

PubMed1996

Identification and analysis of an Autographa californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus envelope: ODV-E56.

An Autographa californica nuclear polyhedrosis virus gene encoding an occlusion-derived virus (ODV) envelope protein of 56 kDa was identified and sequenced. Transcription initiates from a conserved baculovirus late motif (ATAAG) with transcripts detected from 16 through 72 hr p.i. The protein is detected in infected cell extracts from 36 hr p.i. Western blot assay of ODV, BV, viral envelope, and nucleocapsid preparations coupled with immunoelectron microscopy reveal that this protein localizes to the ODV envelope. This protein is named ODV-E56 to identify its viral origin, envelope location, and apparent molecular weight. ODV-E56 is enriched in viral induced intranuclear microvesicles as determined by immunogold labeling. A mutant was constructed with the C-terminal portion of the protein replaced with beta-galactosidase. The fusion protein, E56-beta-gal, locates to the viral nucleocapsids and not to the ODV envelope or intranuclear microvesicles. This suggests that the signals necessary for transport and/or retention into these structures lies within the C-terminal portion of ODV-E56. Additionally, both ODV-E56 and E56-beta-gal are enriched in electron dense regions that cluster around the inner nuclear membrane and within the nucleoplasm.

Amino Acid Sequence↗

PubMed1994

Autographa californica nuclear polyhedrosis virus, PDV, and ECV viral envelopes and nucleocapsids: structural proteins, antigens, lipid and fatty acid profiles.

Autographa californica nuclear polyhedrosis virus (AcMNPV) infection results in the production of two types of infectious, enveloped viruses. As both of these viral forms play significantly different roles in the virus life cycle, the different functional characteristics in the roles of the virus may be explained, in part, by the protein and lipid composition and source of the viral envelopes. Both viruses utilize different maturation and envelopment strategies: Extracellular virus (ECV) obtains an envelope by budding from the host cell plasma membrane, while the envelope of polyhedra-derived virus (PDV) is obtained within the nucleus of the host cell. There is compelling evidence for differences between ECV and PDV structural proteins; however, no previous study directly compares ECV and PDV purified from the same source and little data are available on the protein and lipid composition of the viral envelopes. This study begins the systematic comparison of ECV, PDV, and their envelopes to target proteins for use as probes to study the molecular and biochemical basis of viral envelopment within the nucleus. AcMNPV ECV and PDV were isolated from infected Spodoptera frugiperda cells and fractionated into their respective envelope and nucleocapsid fractions. The structural protein, glycoprotein, and phosphoprotein composition of ECV, PDV, and their envelope and nucleocapsid fractions are analyzed and compared, and antigens of ECV and PDV viral envelope are identified. A number of structural proteins are different between ECV and PDV. ECV is enriched for proteins at 67, 45, and 35 kDa, while proteins at 89, 70, 60, 50, and 25 kDa are enriched in PDV. At least two proteins, PDV-E66 and PDV-E43, are identified to be specific for the PDV envelope. There are more N-glycosylated proteins in ECV than in PDV, with ECV-specific proteins found at 137, 128, 89, 45, and 40 kDa. PDV glycoproteins are 70, 53, 49, 42, 40, and 31 kDa. Most phosphoproteins of both ECV and PDV are predominantly found in the viral envelopes. The predominant phosphoprotein of ECV is 85 kDa, whereas PDV major phosphoprotein is 36 kDa. This study presents the first report of the phospholipid and fatty acid content of ECV and PDV viral envelopes. The major phospholipid of ECV is phosphatidylserine (50%), while phosphatidylcholine and phosphatidylethanolamine are the major phospholipids of PDV (39 and 30%, respectively). Since PDV is enveloped within the nucleus of the host cell, the PDV phospholipid composition is compared with the phospholipid composition of purified S. frugiperda (Sf9) nuclei and this analysis demonstrates significant differences between these two membrane systems.

Animals↗

PubMed1994

Transcription, translation, and cellular localization of PDV-E66: a structural protein of the PDV envelope of Autographa californica nuclear polyhedrosis virus.

A late gene encoding a 66-kDa structural protein of Autographa californica nuclear polyhedrosis virus was mapped and sequenced (26.9-29.7 MU). Transcription initiates from two conserved TAAG motifs (-15 and -37) with transcripts detected from 12 to 72 hr pi. The protein is detected in infected Spodoptera frugiperda (Sf9) cells from 24 to 72 hr pi. Western-blot and immunoelectron microscopic data identify this protein as specific for the polyhedra-derived virus (PDV) envelope. This protein has been named PDV-E66 to identify its viral origin, envelope location, and apparent molecular weight. In addition to being a structural protein of the PDV envelope, PDV-E66 is enriched in foci of microvesicles in the nuclei of infected Sf9 cells.

Amino Acid Sequence↗

PubMed1992

Sequence, genomic organization of the EcoRI-A fragment of Autographa californica nuclear polyhedrosis virus, and identification of a viral-encoded protein resembling the outer capsid protein VP8 of rotavirus.

We present the sequence and genomic organization of the EcoRI-A fragment of the Autographa californica multicapsid nuclear polyhedrosis virus, which represents 11% of the AcMNPV genome. Fifteen putative open reading frames and their respective amino acid sequences are described. One open reading frame is similar to the VP8 protein of rotavirus.

Amino Acid Sequence↗

PubMed1990

The metalloprotease gene of Serratia marcescens strain SM6.

Utilizing the DNA sequence of the metalloprotease from Serratia strain E-15, we isolated and sequenced the homologous gene from Serratia strain SM6. These two genes are similar at both the DNA and protein sequence level. Expression of the protease gene in Escherichia coli was achieved by use of the lac promoter. This resulted in the production and excretion of an immunologically detectable but inactive protein of slightly higher molecular weight than that from Serratia. We introduced the cloned gene into previously described protease mutants. The observed pattern of protease expression suggested that these mutations fall into three classes.

Amino Acid Sequence↗

PubMed1990

Expression of Serratia marcescens extracellular proteins requires recA.

A previously described regulatory mutation which abolishes expression of the extracellular nuclease of Serratia marcescens is shown to be a mutation of the Serratia recA gene. The defect in nuclease expression could be restored by introducing a plasmid carrying the recA gene of Escherichia coli. The DNA sequence of the Serratia gene is very similar to that of the E. coli gene. The putative LexA-binding site of the Serratia recA gene is almost identical to that of E. coli, along with the promoter. A similar LexA-binding site can also be found upstream of the nuclease gene. As expected from this finding, we show that nuclease expression can be induced by SOS-inducing agents such as mitomycin C. Although inducible in S. marcescens, the nuclease was expressed only at the uninduced levels in E. coli and could not be induced by mitomycin C. The extracellular chitinase and lipase were similarly affected by the mutations altering nuclease expression and were also induced by mitomycin C.

Bacterial Proteins↗

PubMed1988

Retinal pigment epithelial cell plasma membrane: a monoclonal antibody study.

The plasma membranes of retinal pigment epithelial cells are highly specialized organelles with multiple functions including nutritional and metabolic support of the photoreceptor cells. Using purified bovine retinal epithelial cell plasma membranes as antigen, we produced two monoclonal antibodies, MAbD1-C6 and MAbD1-C8, that cross react with the plasma membranes from bovine, rat and human retinal pigment epithelial cells. In radioimmunoassay both MAbD1-C6 and MAbD1-C8 had similar affinities for bovine plasma membranes. Both monoclonal antibodies identified a protein of 72 Kd with an apparent subunit of 32-35 Kd. The protein was localized to the cell surface of human and bovine retinal pigment epithelium by immunocytohistochemistry. In the normal eye the antigen identified by the monoclonal antibodies was strongly associated with the retinal pigment epithelium and weakly associated with lens tissue. Using either monoclonal antibody, components of purified bovine or rat retinal pigment epithelial plasma membranes were precipitated from solution. Based on these results, we conclude that both monoclonal antibodies are closely related and that they may be useful for the isolation and study of retinal pigment epithelial cell structure.

Animals↗

PubMed1988

The potential role of adenosine in regulating blood flow in the eye.

Blood flow in the eye has shown a remarkable ability to autoregulate regardless of intraocular pressure, perfusion pressure or alterations in arterial pressure. This study investigates the possibility that adenosine may play a role in regulating ocular blood flow. Ocular blood flow was measured using radio-labelled 85Sr microsphere and laser Doppler techniques. When two adenosine uptake inhibitors, dipyridamole and papaverine were injected intravitreally, the ocular blood flow increased in all ocular tissues tested: iris, iris root-ciliary body, retina and choroid. This increase in blood flow was blocked by the addition of the adenosine antagonist, 8-phenyltheophylline. The increase in flow produced by dipyridamole continued for up to an hour after administration. The increase in blood flow in individual ocular tissues does not appear to be due to shunting (ie. redistribution) of flow because total blood flow to the eye increased but the percent total flow to each individual tissue remained near control values.

Adenosine↗

PubMed1988

Characterization of pigment epithelial cell plasma membranes from normal and dystrophic rats.

Retinal pigment epithelial cell plasma membranes were isolated from the eyes of normal and RCS-dystrophic rats by binding glass microbeads to the intact pigment epithelial cell layer, removal of the bead-bound cells from the eyes and subsequent sucrose density gradient centrifugation. Plasma membranes were recovered from the gradients in identical yields and characterized by membrane marker enzymes, lipid analysis and SDS-polyacrylamide gel electrophoresis. Membrane purification by alkaline phosphodiesterase I and 5'nucleotidase activities averaged 8-fold for normal rats and 5.5 for the dystrophic rats. The ratio of cholesterol per microgram protein indicated 6 to 7-fold purification for both types of plasma membranes. Na+K+-ATPase in the normal and mutant rat plasma membranes was purified 5- and 3.5-fold, respectively, but the specific activities of both Na+K+-ATPase and 5'nucleotidase were higher in the dystrophic rat membranes than in normal. Subcellular organelle contamination was low and relatively uniform in both types of membranes, while opsin contamination was less than 1%. By electrophoretic analysis the plasma membrane proteins were similar, with 30-40 identifiable bands present in each membrane type. The plasma membranes both contain high levels of cholesterol, sphingomyelin and phosphatidylcholine and low levels of polyunsaturated fatty acids. However, the dystrophic rat membranes had significantly higher levels of docosahexaenoic acid than normal, and significantly lower levels of arachidonic acid. The differences in these plasma membrane fatty acids and in the membrane-bound enzymes may affect the ionic balance of the interphotoreceptor matrix or otherwise contribute to degenerative changes in dystrophic rat photoreceptors.

Animals↗