Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes.

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.

Cytotoxic T-lymphocyte epitope immunodominance in the control of choroid plexus tumors in simian virus 40 large T antigen transgenic mice.

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.

Context-dependent immunogenicity of an S206G-substituted H-2Db-restricted simian virus 40 large T antigen epitope I variant.

SV40 large tumor Ag (Tag) contains four H-2b-restricted (I, II/III, IV, and V) CTL epitopes. A hierarchy exists among these CTL epitopes. CTL directed against epitopes I, II/III, and IV are readily detected following immunization of H-2b mice with SV40, Tag-transformed syngeneic cells, or a vaccinia recombinant that expresses full-length Tag, while epitope V-specific CTL are not. The mechanisms that define this hierarchy remain unknown. Initial studies have shown that the locations of epitopes I and V within SV40 Tag do not determine the immunological potencies of these epitopes. Like the wild-type Tag, derivatives in which the locations of epitopes I and V were precisely reversed within Tag failed to induce epitope V-specific CTL, but did induce epitope I-specific CTL. The use of an S206G-substituted epitope I variant (GAINNYAQKL) revealed that the S206G variant sequence induced CTL when located within the native epitope I context, but failed to do so when located within the epitope V context of Tag. Mutagenesis of residues adjacent to the S206G-substituted epitope I variant revealed that the identity of the residue flanking the amino terminus of the S206G variant was critical when it resided within the epitope V location, but not within the epitope I location. These results demonstrate that effects imposed by both regional context and adjacent residues can modulate immunogenicity, but that the relative importance of such effects varies in an epitope-dependent manner.

Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity.

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.

A molecular basis for how a single TCR interfaces multiple ligands.

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.

Role of a subdominant H-2Kd-restricted SV40 tumor antigen cytotoxic T lymphocyte epitope in tumor rejection.

SV40-transformed mKSA cells (H-2d) readily induce progressively growing tumors in adult syngeneic BALB/c mice while expressing the full complement of H-2d MHC class I antigens. BALB/c mice previously immunized with SV40, soluble SV40 T antigen, or irradiated SV40-transformed syngeneic, allogeneic, or xenogeneic cells reject an mKSA tumor challenge even though these mice have been considered low- or nonresponders to T antigen due to difficulty in demonstrating SV40 T antigen-specific CTL. We have investigated the role of H-2d-restricted CTL in the rejection of SV40 tumors in BALB/c mice. Immunization of BALB/c mice with SV40 induced T antigen-specific CTL which were largely. H-2Ld-restricted. However, following repeated in vitro restimulation with mKSA cells, CTL emerged which recognized a subdominant H-2Kd-restricted epitope corresponding to T antigen residues 499-507. Immunization of BALB/c mice with a recombinant vaccinia virus expressing the T499-507 epitope provided partial protection against a challenge of syngeneic mKSA tumor cells and induced the generation of T499-507-specific CTL. These results indicate that a subdominant H-2Kd-restricted CTL epitope can participate in the rejection of SV40 tumors in BALB/c mice.

An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope.

An immunological hierarchy among three H-2Db-restricted cytotoxic T lymphocyte (CTL) determinants in simian virus 40 (SV40) large T antigen (Tag) was described previously: determinants I and II/III are immunodominant, whereas determinant V is immunorecessive. To assess the immunogenicity of each determinant individually and define mechanisms that contribute to the immunorecessive nature of determinant V, we constructed a panel of recombinant vaccinia viruses (rVVs) expressing minigenes encoding these determinants in various polypeptide contexts. We found the following. (i) Immunization of mice with an rVV encoding full-length SV40 Tag resulted in priming for CTL responses to determinants I and II/III but not determinant V. (ii) rVVs encoding peptide I or II/III in the cytosol or targeted to the endoplasmic reticulum (ER) were highly antigenic and immunogenic. (iii) rVVs encoding peptide V minigenes were antigenic and immunogenic if the peptide was targeted to the ER, expressed in the cytosol with short flanking sequences, or expressed from within a self-protein, murine dihydrofolate reductase. (iv) Presentation of the nonflanked peptide V (preceded by a Met codon only) could be enhanced by using a potent inhibitor of the proteasome. (v) H-2Db-epitope V peptide complexes decayed more rapidly than complexes containing epitope I or II/III peptides. In brefeldin A blocking experiments, functional epitope V complexes were detected longer on targets expressing ER-targeted epitope V than on targets expressing forms of epitope V dependent on the transporter associated with antigen processing. Therefore, limited formation of relatively unstable cell surface H-2Db complexes most likely contributes to the immunorecessive nature of epitope V within SV40 Tag. Increasing the delivery of epitope V peptide to the major histocompatibility complex class I presentation pathway by ER targeting dramatically enhanced the immunogenicity of epitope V.

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.

Functional analysis of amino acid residues encompassing and surrounding two neighboring H-2Db-restricted cytotoxic T-lymphocyte epitopes in simian virus 40 tumor antigen.

Simian virus 40 tumor (T) antigen contains three H-2Db-and one H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes (sites). Two of the H-2Db-restricted CTL epitopes, I and II/III, are separated by 7 amino acids in the amino-terminal one third of T antigen. In this study, we determine if the amino acids separating these two H-2Db-restricted CTL epitopes are dispensable for efficient processing and presentation. In addition, the importance of amino acid residues lying within and flanking the H-2Db-restricted epitopes I and II/III for efficient processing, presentation, and recognition by site-specific CTL clones was determined by using T-antigen mutants containing single-amino-acid substitutions between residues 200 and 239. Using synthetic peptides in CTL lysis and major histocompatibility complex class I stabilization assays, CTL recognition site I has been redefined to include residues 206 to 215. Substitutions in amino acids flanking either site I or site II/III did not affect recognition by any of the T-antigen-specific CTL clones. Additionally, the removal of the 7 residues separating site I and site II/III did not affect CTL recognition, thus demonstrating that these two epitopes when arranged in tandem in the native T antigen can be efficiently processed and presented to CTL clones. Differences in fine specificities of two CTL clones which recognize the same epitope (Y-1 and K-11 for site I and Y-2 and Y-3 for site II/III) have been used in conjunction with synthetic peptide variants to assign roles for residues within epitopes I and II/III with respect to TCR recognition and/or peptide-major histocompatibility complex association.

Cytotoxic T lymphocyte escape variants, induced mutations, and synthetic peptides define a dominant H-2Kb-restricted determinant in simian virus 40 tumor antigen.

Immunization of C57BL/6 mice with syngeneic cells transformed by simian virus 40 large T antigen (SV40 T ag) induces the generation of T antigen-specific cytotoxic T lymphocytes (CTL) which are restricted by the major histocompatibility class I antigens H-2Db and H-2Kb. Previous studies have shown that the H-2Db-restricted CTL response is directed to at least three distinct epitopes (I, II/III, and V) in the SV40 T antigen which have been precisely mapped using deletion mutagenesis and overlapping synthetic peptides. Although in vivo the CTL response to SV40 T antigen is dominated by the H-2Kb class I antigen, the precise location of the H-2Kb-restricted epitope(s) was not known, and whether there was multiplicity of H-2Kb-restricted epitopes remained unclear. In this study, we have defined the minimal recognition epitope for the SV40-specific H-2Kb-restricted CTL clone Y-4 as T antigen residues 404-411 by using T antigen deletion and point mutants and synthetic peptides. DNA sequence analysis of the region encoding residues 404-411 from the T antigens expressed in three independently isolated CTL clone Y-4 escape variants identified inactivating mutations capable of abrogating CTL recognition. Estimation of CTL precursor (CTLp) frequencies by limiting dilution analysis revealed that CTLp specific for epitope IV represent a large percentage of the total CTL response elicited by the intact T antigen in H-2b mice. Immunization of B6 mice with cells expressing a T antigen derivative deleted of residues 404-411 revealed that site IV represents the only immunodominant H-2Kb-restricted epitope within T antigen.

SIP1 is a catabolite repression-specific negative regulator of GAL gene expression.

The yeast Snf1p kinase is required for normal expression of many genes involved in utilization of non-glucose carbon. Snf1p is known to associate with several proteins. One is Sip1p, a protein that becomes phosphorylated in the presence of Snf1p and thus is a candidate Snf1p kinase substrate. We have isolated the SIP1 gene as a multicopy suppressor of the gal83-associated defect in glucose repression of GAL gene expression. Multicopy SIP1 also suppressed the gal82-associated defect in glucose repression, suggesting that SIP1, GAL83 and GAL82 function interdependently. Multicopy SIP1 gene reduces GAL1, GAL2, GAL7 and GAL10 gene expression three- to fourfold in cells grown in the presence of glucose but has no effect in cells grown on nonrepressing carbon. Sip1-deletion cells exhibited a two- to threefold increase in GAL gene expression compared to wild-type cells when grown on glucose. These studies show that SIP1 is a catabolite repression-specific negative regulator of GAL gene expression. Northern analysis revealed two SIP1 transcripts whose relative abundance changed with carbon source. Western blots revealed that Sip1p abundance is not markedly affected by carbon source, suggesting that Sip1p may be regulated post-translationally.

Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests functional domains.

Efficient transcription of many Saccharomyces cerevisiae genes requires the GAL11 Protein. GAL11 belongs to a class of transcription activator that lacks a DNA-binding domain. Such proteins are thought to activate specific genes by complexing with DNA-bound proteins. To begin to understand the domain structure-function relationships of GAL11 we cloned and sequenced a homologue from the yeast Kluyveromyces lactis, Kl-GAL11. The two predicted GAL11 proteins show high overall amino acid conservation and an unusual amino acid composition including 18% glutamine, 10% asparagine (S. cerevisiae) or 7% (K. lactis), and 8% proline (K. lactis) or 5% (S. cerevisiae) residues. Both proteins have runs of pure glutamines. Sc-GAL11 has glutamine-alanine runs but in Kl-GAL11 the alanines in such runs are replaced by proline and other residues. The primary sequence similarity is reflected in functional similarity since a gal11 mutation in K. lactis creates phenotypes similar to those seen previously in gal11-defective S. cerevisiae. In addition, Kl-GAL11 complements a gal11-defect in S. cerevisiae by partially restoring induction of GAL1 expression, growth on nonfermentable carbon sources, and phosphorylation of GAL4.

GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator.

The GAL4 protein of Saccharomyces cerevisiae is a DNA-binding transcriptional activator that is highly specific for the GAL genes. In vivo levels of GAL gene transcription are closely correlated with the phosphorylation state of GAL4. In vivo levels of GAL gene transcription are also affected by the activity of the GAL11 (SPT13) protein, a protein that has been implicated as a global auxiliary transcriptional factor. Here we examine the influence of GAL11 (SPT13) on the phosphorylation state of GAL4. Cells bearing a gal11 deletion mutation are defective in the production or maintenance of GAL4III, a phosphorylated form of GAL4 that is associated with higher levels of GAL gene transcription. In addition, the gal11 deletion cells are reduced in total GAL4 protein. However, the fivefold-reduced expression of the GAL1 gene observed in gal11 deletion cells cannot be due solely to reduced levels of total GAL4 protein, since gal11 deletion cells amplified for GAL4 production are still markedly reduced in GAL4 protein-dependent transcription. Thus, these data demonstrate that the GAL11 protein augments GAL4 protein-dependent transcription in a manner that is tightly coupled to the formation or maintenance of a phosphorylated form of GAL4.

Phosphorylated forms of GAL4 are correlated with ability to activate transcription.

GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.

Regulated phosphorylation and dephosphorylation of GAL4, a transcriptional activator.

In yeast, galactose triggers a rapid GAL4-dependent induction of galactose/melibiose regulon (GAL/MEL) gene transcription, and glucose represses this activation. We discovered that alterations in the physical state of the GAL4 protein correlate with activation and repression of the GAL/MEL genes. Using Western immunoblot assay, we observe two electrophoretic forms of GAL4 protein-GAL4I and GAL4II-in noninduced cells. In the absence of glucose, the addition of galactose to such cells results in the rapid appearance of a third and slower-migrating form, GAL4III, which differs from at least GAL4I by phosphorylation. GAL80-deletion cells that constitutively transcribe galactose-responsive genes due to the lack of the GAL80 protein, an antagonist of the GAL4 protein, exhibit GAL4III without galactose addition. Addition of glucose, which results in rapid repression of galactose gene transcription, triggers a rapid elimination of GAL4III and an increase in GAL4II. Cycloheximide experiments provide evidence that the galactose- and glucose-triggered GAL4 protein mobility shifts are due to post-translational modification. GAL4III is labeled with [32P]phosphate in vivo; in vivo 35S-labeled GAL4III could be converted by phosphatase treatment in vitro to GAL4I. We present a model proposing that phosphorylation state changes in the GAL4 protein are key to modulating its activity.

Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast.

High-level, galactose-inducible expression originating from GAL promoters in Saccharomyces cerevisiae is mediated by highly specific interactions between the GAL4-coded protein and nucleotide sequences. The potential utility of recombinant GAL promoter/foreign gene constructions for the regulatable and high-level expression of foreign proteins in yeast is well recognized. However, the utility of this system is limited severely in the case of multiple copies of such constructions due to the very low level of the GAL4-coded protein and to the loss of inducibility which occurs if levels of the GAL4 protein are amplified constitutively. To surmount these limitations, we have constructed a novel yeast strain which overproduces the GAL4 protein in a regulated fashion. This 'integrant' strain contains an integrated copy of a hybrid gene consisting of the galactose-inducible GAL10 promoter fused to the GAL4 structural gene. In the absence of galactose, the integrant strain and the isogenic 'non-integrant' parental strain show only a basal level of transcription from the constitutively active chromosomal GAL4 gene. However, following the addition of galactose to the culture medium, the 'integrant' strain synthesizes at least 20-fold more GAL4 mRNA and substantially more GAL4 protein than the 'non-integrant' strain. A high-copy-number expression vector containing the GAL10 promoter and alpha mating factor pre-pro leader fused to the structural gene for Epstein-Barr virus gp350 was introduced into both types of cells. The resulting transformed 'integrant' cells produced approximately five-fold more gp350 mRNA and ten-fold more gp350-related proteins than the transformed 'non-integrant' cells following galactose induction. This 'integrant' strain should prove generally useful for the maximal, regulated expression in yeast of structural genes driven by a galactose-inducible promoter.

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.

Species-dependent variations in erythrocyte membrane skeletal proteins.

Two mammalian species (porcine and murine) have erythrocytes that are being widely used to study membrane protein synthesis and red cell aging. Erythrocytes of these species however, are significantly smaller than those of the human. Before results obtained from study of these red cells can be applied to human cells, the membrane skeleton of these species must be investigated to determine if the skeletal elements are equivalent. Both pig and mouse bands 4.1b were of lower molecular weight than human 4.1b, and the a/b ratio was lower. In each species, 4.1a and b were sequence-related phosphoproteins, and yielded substantially different one-dimensional peptide maps. Band 3 of pig and mouse erythrocytes had a higher molecular weight than human band 3 and also had differing one-dimensional peptide maps after limited proteolytic cleavage with three different enzymes. In each species, free band 3 and band 3 bound to the membrane skeleton had identical peptide maps. Other major membrane skeletal components (spectrin, actin, and bands 2.1 and 4.2) seem to be very similar in molecular weight in various species. These results demonstrate that the molecular weights and relative proportions of the membrane skeletal elements are species dependent.