Hydrophilic Polypeptide Research Articles

The Saccharomyces cerevisiae VMA6 gene encodes the 36-kDa subunit of the vacuolar H(+)-ATPase membrane sector

The yeast vacuolar membrane proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral catalytic, and integral membrane domains. At least eight proteins cofractionate with purified preparations of the enzyme including 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa polypeptides (Kane, P.M., Yamashiro, C.T., and Stevens, T.H. (1989a) J. Biol. Chem. 264, 19236-19244). We took a reverse genetic approach to clone the structural gene for the 36-kDa subunit of the V-ATPase, VMA6, vma6 null mutants displayed growth characteristics typical of other vma mutants including sensitivity to media buffered at neutral pH or media containing 100 mM Ca2+. Vacuolar acidification was defective in vma6 cells and isolated vacuolar membrane preparations contained no detectable V-ATPase activity. The VMA6 gene encodes a hydrophilic polypeptide of 345 amino acids (predicted molecular mass 39.8-kDa). We present evidence that the VMA6 gene product (Vma6p) is a non-integral membrane component of the membrane pore domain and is required for V-ATPase complex assembly. Vma6p was removed from wild type vacuolar membranes by strong chaotropic agents such as alkaline Na2CO3 or 5M urea, which did not remove integral membrane polypeptides. In yeast cells lacking the integral membrane portion of the V-ATPase complex, Vma6p was unable to stably associate with vacuolar membranes. Conversely, in mutants specifically lacking Vma6p, components of the V-ATPase integral membrane domain were destabilized, and peripheral subunits failed to assemble onto vacuolar membranes. These results are discussed in the context of a developing model for V-ATPase assembly in yeast.

The complete general secretory pathway in gram-negative bacteria

The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins.

Differential expression of two related, low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh

Plant cold acclimation is correlated to expression of low-temperature-induced (lti) genes. By using a previously characterized lti cDNA clone as a probe we isolated a genomic fragment that carried two closely located lti genes of Arabidopsis thaliana. The genes were structurally related with the coding regions interrupted by three similarly located short introns and were transcribed in the same direction. The nucleotide sequences of the two genes, lti78 and lti65, predict novel hydrophilic polypeptides with molecular weights of 77,856 and 64,510, respectively, lti78 corresponding to the cDNA probe. Of the 710 amino acids of LTI78 and 600 amino acids of LTI65, 346 amino acids were identical between the polypeptides, which suggests that the genes may have a common origin. Both lti78 and lti65 were induced by low temperature, exogenous abscisic acid (ABA) and drought, but the responsiveness of the genes to these stimuli was markedly different. Both the levels and the temporal pattern of expression differed between the genes. Expression of lti78 was mainly responsive to low temperature, that of lti65 to drought and ABA. In contrast to the induction of lti78, which follows separate signal pathways during low-temperature, ABA and drought treatment, the drought induction of lti65 is ABA-dependent and the low-temperature induction appears to be coupled to the ABA biosynthetic pathway. This differential expression of two related genes may indicate that they have somewhat different roles in the stress response.

The complete general secretory pathway in gram-negative bacteria.

The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins.

Characterization of a Cold-Regulated Wheat Gene Related to Arabidopsis cor47

The cDNA clone pWG1 represents a gene, designated cor39, from Triticum aestivum L. cv Winoka (a winter wheat) that is related to a cold-regulated Arabidopsis thaliana L. (Heyn) gene, cor47. In vitro transcription/translation experiments in conjunction with DNA sequence analysis indicated that cor39 encodes a hydrophilic polypeptide of 39 kD (isoelectric point of 7.5), designated COR39. The polypeptide is composed primarily of two sequences, each of which is repeated six times. One sequence, which is lysine rich, occurs in COR47 (the polypeptide encoded by Arabidopsis cor47) and group II LEA proteins, polypeptides hypothesized to have roles in desiccation and drought tolerance (J. Baker, C. Steele, L. Dure III [1988] Plant Mol Biol 11: 277-291). The second sequence, which is glycine rich, occurs in some, but not all, group II LEA proteins. Southern analysis indicated that wheat has a number of loci related to cor39. Transcripts of about 3.3, 1.5, and 0.8 kb that hybridize with cor39 were found to accumulate in leaf, root, and crown tissues of cold-acclimated plants; they accumulated rapidly in response to low temperature and returned quickly to low levels when plants were returned to normal growth temperature. Transcripts hybridizing with cor39 were present at relatively high levels in wheat seeds and accumulated in plants in response to exogenous application of ABA and water stress. The similarities in expression of wheat and Arabidopsis cor genes and possible functional relationships among COR39, COR47, and LEA proteins are discussed.

Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. II. VAM7, a gene for regulating morphogenic assembly of the vacuoles.

VAM7 gene function has shown to be required for proper morphogenesis of the vacuole in yeast. The DNA fragments that complemented the defective vacuolar morphology of the vam7-1 mutation were isolated from a yeast genomic library. An overlapping 2.5-kilobase BglII-HindIII region was found to be sufficient for complementation of the vam7-1 phenotype. This fragment was integrated at the chromosomal VAM7 locus, indicating that it contained an authentic VAM7 gene. On nucleotide sequencing of the VAM7 gene, an open reading frame of 948 base pairs, coding for a hydrophilic polypeptide of 316 amino acid residues, was identified. The deduced amino acid sequence of the carboxyl-terminal region of the VAM7 gene product has heptad repeats and shows potential ability to form a coiled-coil structure. Disruption of VAM7 was not lethal; cells with a disrupted VAM7 gene did not, however, have a prominent large vacuoles but rather numerous small compartments that accumulated the histochemical marker molecule of the vacuolar compartment. They contained mature forms of the vacuolar marker proteins carboxypeptidase Y and vacuolar glycoprotein vgp72. A mutant with both vam7 and vam5 null mutations was constructed and shown to have neither vacuolar structures stained with ade-related fluorochrome nor mature forms of vacuolar marker proteins. These findings suggested that the VAM7 gene product functions in the process of morphogenic assembly of the vacuolar compartment but is not involved in the protein sorting and delivery to the vacuole.

DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide

Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the "boiling-stable" polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed.

POLYPEPTIDES WITH ENHANCED FOAM POTENTIAL

The ability of beer to produce a good foam is strongly influenced by the level of foam active polypeptides. It has previously been proposed that a means of ensuring an adequate level of such species is to add an exogenous preparation of foam active protein3. One such preparation, hydrolysed liquid egg white (HLEW), has been shown to impart a good foam to beer with substandard foam performance, without detriment to product quality. The foam active properties of this material are the subject of the work described here. HLEW was characterised by a combination of reverse phase chromatography and polyacrylamide gel electrophoresis to reveal a heterogeneous mixture of low molecular weight (2100–6000 daltons) relatively hydrophilic polypeptides. When this material was subjected to foaming and the foam positive and foam negative fractions similarly characterised, it was apparent that the foam positive fraction consisted almost exclusively of hydrophobic polypeptides. Accordingly, preparative reverse phase chromatography was used to isolate foam active fractions from the hydrophobic species present. These fractions were subsequently subjected to both functional and physical characterisation. It was apparent from a small-scale Rudin test that the foam activity per unit dry weight of protein was enhanced in the foam active factions; in one case an enhancement of 2 fold greater than the HLEW itself was observed. Moreover, upon addition to beer a positive effect on HRV was achieved with as little as 0.0025 mg protein/ml beer. Physical characterisation of the foam active material revealed the presence of tightly bound polypeptide aggregates which could only be separated by the use of protein denaturing agents.

Revised nucleotide sequence of the gltP gene, which encodes the proton-glutamate-aspartate transport protein of Escherichia coli K-12.

The gene encoding the proton-glutamate carrier (GltP) of Escherichia coli K-12 was sequenced, and the primary structure of the protein was analyzed. The nucleotide sequence was found to differ in several aspects from the previously published sequence (B. Wallace, Y. Yang, J. Hong, and D. Lum, J. Bacteriol. 172:3214-3220, 1990). The corrected open reading frame encodes a protein of 437 (instead of 395) amino acids. Hydropathy analysis predicts 12 membrane-spanning alpha-helical regions. The complementary strand does contain an open reading frame possibly encoding a highly hydrophilic polypeptide of 272 amino acids.

CDNA sequence analysis and expression of two cold-regulated genes ofArabidopsis thaliana

The DNA sequences of cDNAs for two cor (cold-regulated) genes of Arabidopsis thaliana L. (Heyn) were determined. One cDNA (approximately 70% full-length) corresponds to a cor gene, designated cor47, that encodes a 47 kDa hydrophilic polypeptide. The data indicate that COR47 has amino acid sequence homology with Group II LEA (late embryogenesis abundant) proteins, a class of proteins that accumulate late in embryo development. DNA sequence analysis of a second cDNA (containing the complete protein coding sequence) indicates that it represents a cor gene, designated cor6.6, that encodes an alanine-rich 6.6 kDa hydrophilic polypeptide. COR6.6 is almost identical to KIN1, a cold-regulated Arabidopsis gene that has been suggested to have amino acid sequence similarities with type I fish antifreeze proteins (S. Kurkela, M. Franck, Plant Mol Biol 15: 137-144, 1990). Northern analysis indicated that transcripts for cor47 and cor6.6 do not accumulate to high levels in late-developing embryos or fresh mature seeds as is typical of lea gene transcripts. The similarities and differences between COR and LEA proteins are discussed as are their possible roles in freezing and drought tolerance.

Biomembrane Model from Stable Polymer Membrane Having Polypeptide Domains as Channels

Abstract A novel polymer membrane having a transmembrane permeation pathway (channel) was prepared from a polyvinyl-polypeptide graft copolymer. The transmembrane continuous phases of the hydrophilic polypeptide were found to be formed in the stable matrix from vinyl polymer and to function as a permeation pathway for polar substances; the polypeptide domain is regarded as a membrane protein model. The infrared and circular dichroism spectra of the membrane showed the pH-dependent conformational change of the polypeptide segment. Regulation of permeability and permselectivity was performed by pH based on the conformational transition of the channel-composing polypeptide. In addition, the membrane has been found to respond to divalent cations, cationic surfactants, urea, and organic solvents in terms of membrane permeability as well as conformational status of polypeptide.

Novel purification of the catalytic domain of Golgi alpha-mannosidase II. Characterization and comparison with the intact enzyme.

Rat liver alpha-mannosidase II, a hydrolase involved in the processing of asparagine-linked oligosaccharides, is an integral membrane glycoprotein facing the lumen of Golgi membranes. We have previously shown (Moremen, K. W., and Touster, O. (1986) J. Biol. Chem. 261, 10945-10951) that mild chymotrypsin digestion of permeabilized or solubilized Golgi membranes will result in the cleavage of the intact 124,000-dalton alpha-mannosidase II subunit, releasing a 110,000-dalton hydrophilic polypeptide which contains the catalytic site. Consistent with the removal of a membrane binding domain, the chymotrypsin-generated 110,000-dalton peptide was found exclusively in the aqueous phase in Triton X-114 phase separation studies, whereas the intact enzyme was found in the detergent phase. Taking advantage of this conversion in phase partitioning behavior, a purification procedure was developed to isolate the 110,000-dalton proteolytic digestion product as a homogeneous polypeptide for further characterization and protein sequencing at a yield of greater than 65% from a rat liver Golgi-enriched membrane fraction. An improved purification procedure for the intact enzyme was also developed. The two forms of the enzyme were compared yielding the following results. (a) The catalytic activity of the intact and cleaved forms of alpha-mannosidase II were indistinguishable in Km, Vmax, inhibition by the alkaloid, swainsonine, and in their activity toward the natural substrate GlcNAc-Man5GlcNAc. (b) Both the intact and cleaved forms of the enzyme appear to be disulfide-linked dimers. (c) The two forms of the enzyme contain different NH2-terminal sequences suggesting that the cleaved NH2 terminus contains the membrane-spanning domain. (d) Additional peptide sequences were obtained from proteolytic fragments and cyanogen bromide digestion products in order to create a partial protein sequence map of the enzyme. These results are consistent with a model common among Golgi processing enzymes of a hydrophilic catalytic domain anchored to the lumenal face of Golgi membranes through an NH2-terminal hydrophobic membrane-anchoring domain.

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.

Identification of a low- Mr acidic nuclear protein as prothymosin α

We have purified to homogeneity a 15-kDa perchloric acid (PCA)-soluble protein from rat thymus nuclei. This highly acidic protein showed a M r, of ca. 30 kDa in acetic acid/urea gels, probably due to oligomer formation. Sequence analysis of internal tryptic and thennolytic peptides revealed that the purified protein is, in fact, prothymosin α, a very hydrophilic polypeptide, which has been previously classified as a thymic or immunomodulating hormone. We found that prothymosin α is a rather abundant nuclear protein in rat thymus; its concentration is comparable to that of a well-characterized nonhistone protein HMG-14. The subcellular localization and physicochemical properties of prothymosin α suggest that its function is related to those of other long polyacidic regions containing nuclear proteins.

The 31-kDa polypeptide is an essential subunit of the vacuolar ATPase in Saccharomyces cerevisiae.

The VMA4 gene encodes a 26.6-kDa hydrophilic polypeptide which exhibits 34% sequence identity with the E subunit of the vacuolar ATPase from bovine kidney microsomes. The chromosomal VMA4 gene was inactivated by a 171-base pair deletion followed by insertion of the URA3 gene within the coding sequence. Null vma4 haploid mutants are viable. However, their growth is considerably slowed down specially in non-acidic conditions; they are cold sensitive and thermo-sensitive, exhibit poor growth on glycerol medium, and do not accumulate in their vacuole the red pigment of ade2 strains. No bafilomycin-sensitive ATPase is detected in a vacuolar fraction. These properties shared by null mutants in the A, B, and C subunits of the vacuolar ATPase show that the VMA4 polypeptide is also an essential component of the vacuolar ATPase which has been conserved from yeast to mammals. The tightly linked VMA4 and MIP1 (encoding the mitochondrial DNA polymerase) genes are divergently transcribed from face-to-face promoters. About 250 base pairs upstream of the VMA4 gene, Homoll and RPG consensus for the binding of TUF (RAP/GRF1) protein are present, suggesting that the VMA4 gene belongs to this large family of genes involved in cellular growth and division whose transcription is regulated by the TUF protein.

Nature of forces stabilizing the transmembrane protein bacteriorhodopsin in purple membrane

Analysis of the far-ultraviolet solution and the oriented-film circular dichroic (CD) spectra of the purple membrane (PM) has indicated that the alpha-helical segments of its sole protein bacteriorhodopsin (bR) can undergo a significant tilting from the normal to the membrane plane during light-dependent hydroxylamine-mediated bleaching of the bR. However, this drastic change in tertiary structure is free of any observable secondary structural changes. This phenomenon can provide an excellent means for studying the relative contributions of forces responsible for the stability of this transmembrane protein within the membrane bilayer. Perturbation of the PM by varying degrees of papain digestion (resulting in changes in the bR ranging from only an elimination of the long COOH-terminal tail to the additional eliminations of the short NH(2)-terminal tail and a number of linkage amino acids between the helical segments of the bR) and by chemical cross-linking with dimethyl adipimidate (resulting primarily in the formation of intramolecular cross-links) resulted in a significant increase in this bleaching-induced tilting in all cases except the one in which only the COOH-tail was eliminated. The most severe perturbation (2-wk papain digestion) increased the net tilt angle per segment from 24 to 39 degrees with no indication of any secondary structural changes. Although these perturbations drastically reduced the structural stability of the bR to bleaching, they caused virtually no observable changes in the intramolecular structure of the bR or the supramolecular structure of the PM based on analysis of extensive absorption, linear dichroic, and CD spectra. In addition, study of the bleaching rates for the perturbed PM samples indicated that a linear correlation exists between the calculated initial bleaching rates and the net tilt angles.Considering the forces generally assumed to account for the stability of transmembrane proteins in membranes, (a) intersegmental hydrogen bonding and electrostatic interactions, (b) electrostatic interactions between hydrophilic polypeptide segments extending outside the bilayer and the many charged lipid heads of the bilayer, and (c) hydrophobic interactions, it is clear that the results of the bleaching experiments eliminate all but perhaps the last as contributing significantly to the bR stability in the PM. Furthermore, they provide more compelling evidence than previously available that the bR is capable of undergoing relatively large retinyldiene-controlled tertiary structural changes and that the chromophoric retinal serves as the most important factor in the native bR structural stability. This dynamic view of the bR bears directly on models proposed for bR function, favoring those in which protein structural metastability, rather than rigidity, is an essential factor. The proteinquake or deformation wave model proposed by this laboratory falls into this category.

The Nucleotide Sequence of Satellite RNA in Grapevine Fanleaf Virus, Strain F13

The nucleotide sequence of cDNA copies of grapevine fanleaf virus (strain F13) satellite RNA has been determined. The primary structure obtained was 1114 nucleotides in length, excluding the poly(A) tail, and contained only one long open reading frame encoding a 341 residue, highly hydrophilic polypeptide of Mr37275. The coding sequence was bordered by a leader of 14 nucleotides and a 3'-terminal non-coding region of 74 nucleotides. No homology has been found with small satellite RNAs associated with other nepoviruses. Two limited homologies of eight nucleotides have been detected between the satellite RNA in grapevine fanleaf virus and those in tomato black ring virus, and a consensus sequence U.G/UGAAAAU/AU/AU/A at the 5' end of nepovirus RNAs is reported. A less extended consensus exists in this region in comovirus and picornavirus RNA.

The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex.

We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.

Attachment and growth of fibroblast cells on polypeptide derivatives.

Attachment and growth of mouse fibroblast cells on polypeptide derivatives with different wettabilities were studied in the presence or absence of serum proteins. In the presence of serum, a peak level of cell attachment was observed for substrates with a contact angle of around 70 degrees. However, no relationship was found between cell attachment and water contact angle of the substrate in the absence of serum. Ca2+ -dependent cell attachment was observed on hydrophobic polypeptides in the absence of serum proteins. This suggests that Ca2+ -dependent membrane proteins function as mediators for cell attachment to hydrophobic polypeptides. In the presence of serum proteins, it was found that cell attachment is affected by the metabolism, Ca2+, and the cytoskeleton of the cell. Cell growth rate on hydrophilic polypeptide substrates was higher than that on intermediate or hydrophobic polypeptide substrates. This demonstrates that the strong interaction between cells and substrates will be unfavorable for a dewebbing process during mitosis.

Benzoxazinone derivatives: new fluorescent probes for two‐color flow cytometry analysis using one excitation wavelength

A new class of fluorescent dye which upon excitation at 488 nm turns red is shown to be probe-suitable for using in flow cytometry alone or in conjunction with fluorescein derivatives. 7-dimethylamino 3-(p-formylstyryl) 1,4 benzoxazin 2-one is suitable for rendering microorganisms, such as Plasmodium merozoites and cells detectable by flow cytometry, allowing a dual fluorescence analysis when the cells are labelled with suitable fluoresceinylated ligands such as fluorescein labeled neoglycoproteins or antibodies. The synthesis of the new benzoxazinone derivatives is described: p-[beta-(7-dimethylamino 1,4 benzoxazin 2-one 3-yl)-vinyl]-phenylpropenoic acid can be easily activated as a hydroxysuccinimide derivative and linked to amino groups of polypeptides. Hydrophilic polypeptides such as poly-L-lysine or glycosylated polymers combined with this new fluorescent dye are shown to be helpful in analyzing cell surface receptors, in dual fluorescence flow cytometry analysis, using a single excitation wavelength and two sets of compounds labeled with the new benzoxazinone derivative and with fluorescein isothiocyanate, respectively. The new benzoxazinone derivative has a high molar absorbance, a good quantum yield fluorescence when it is bound to hydrophilic polypeptides and its fluorescence intensity is not dependent on pH in the physiological pH range.