Soluble Cellular Fraction Research Articles

The roles of integrin-linked kinase in the regulation of myogenic differentiation.

Myogenic differentiation is a highly orchestrated, multistep process that is coordinately regulated by growth factors and cell adhesion. We show here that integrin-linked kinase (ILK), an intracellular integrin- and PINCH-binding serine/threonine protein kinase, is an important regulator of myogenic differentiation. ILK is abundantly expressed in C2C12 myoblasts, both before and after induction of terminal myogenic differentiation. However, a noticeable amount of ILK in the Triton X-100-soluble cellular fractions is significantly reduced during terminal myogenic differentiation, suggesting that ILK is involved in cellular control of myogenic differentiation. To further investigate this, we have overexpressed the wild-type and mutant forms of ILK in C2C12 myoblasts. Overexpression of ILK in the myoblasts inhibited the expression of myogenic proteins (myogenin, MyoD, and myosin heavy chain) and the subsequent formation of multinucleated myotubes. Furthermore, mutations that eliminate either the PINCH-binding or the kinase activity of ILK abolished its ability to inhibit myogenic protein expression and allowed myotube formation. Although overexpression of the ILK mutants is permissive for the initiation of terminal myogenic differentiation, the myotubes derived from myoblasts overexpressing the ILK mutants frequently exhibited an abnormal morphology (giant myotubes containing clustered nuclei), suggesting that ILK functions not only in the initial decision making process, but also in later stages (fusion or maintaining myotube integrity) of myogenic differentiation. Additionally, we show that overexpression of ILK, but not that of the PINCH-binding defective or the kinase-deficient ILK mutants, prevents inactivation of MAP kinase, which is obligatory for the initiation of myogenic differentiation. Finally, inhibition of MAP kinase activation reversed the ILK-induced suppression of myogenic protein expression. Thus, ILK likely influences the initial decision making process of myogenic differentiation by regulation of MAP kinase activation.

Vimentin-dependent utilization of LDL-cholesterol in human adrenal tumor cells is not associated with the level of expression of apoE, sterol carrier protein-2, or caveolin

SW-13 adrenal tumor cells that lack detectable intermediate filaments (IF-free) exhibit an impaired capacity to esterify lipoprotein-derived cholesterol compared with cells that contain vimentin filaments. IF-free cells were found to synthesize and secrete significant amounts of apoE, while apoE secretion was nearly undetectable in cell lines that spontaneously express vimentin. However, stable transfectants that express a mouse vimentin cDNA exhibited elevated levels of cholesterol esterification and apoE secretion compared with untransfected IF-free cells, indicating that apoE secretion is not directly related to the capacity of these cells to esterify cholesterol. Some of the cell lines that differed in the level of apoE synthesis and secretion had similar levels of apoE mRNA, suggesting that the differences in expression involve a post-transcriptional mechanism. Treatment of these cells with forskolin and IBMX, 8br-cAMP, or TPA had no effect on apoE secretion. The level of sterol carrier protein-2 (SCP2) synthesis and the distribution of SCP2 between membrane and soluble cellular fractions was not observably different in cells that contained or lacked vimentin. SW-13 cell lines contained little or no detectable caveolin-1 or caveolin-2.▪ These studies demonstrate that the difference in the capacity of these adrenal tumor cells that contain or lack vimentin filaments to esterify low density lipoprotein-cholesterol is not obviously associated with the level of expression or distribution of apoE, SCP2, or caveolins.—Holwell, T. A., S. C. Schweitzer, M. E. Reyland, and R. M. Evans. Vimentin-dependent utilization of LDL-cholesterol in human adrenal tumor cells is not associated with the level of expression of apoE, sterol carrier protein-2, or caveolin. J. Lipid Res. 1999. 40: 1440–1452.

Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli.

Human SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein containing 14 cysteine residues, was found to partition equally between soluble and insoluble cellular fractions when overexpressed in the Escherichia coli cytoplasm. While the growth temperature and medium pH had little effect on inclusion body formation, co-overproduction of the dnaKJ operon, but not of the groE operon, suppressed aggregation at the expense of intracellular accumulation. Although both forms of the protein were fully reduced in wild-type cells, 70% to 85% of soluble and insoluble SPARC could be converted into oxidized species in a thioredoxin reductase (trxB) null mutant following incubation on ice. Approximately 15% to 20% of SPARC exhibited the electrophoretic mobility of the biologically active protein. Overproduction of the dnaKJ operon in trxB cells decreased the formation of disulfide-bonded SPARC multimers in the aggregated material but not in its soluble counterpart. Our results suggest that the activity responsible for disulfide bond formation in trxB mutants acts at the post-translational level and is able to freely diffuse within inclusion bodies.

Mutagenesis and Chemical Rescue Indicate Residues Involved in β-Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

Phosphatidylethanolamine production and iron homeostasis in Pseudomonas fluorescens

Pseudomonas fluorescens was found to grow in a minimal mineral medium containing citrate, the sole carbon source, complexed to 5 mM iron(III). As the tricarboxylic acid was utilized, dialysis and ultracentrifigation analysis of the spent fluid revealed that iron (III) was associated with phosphatidylethanolamine (PE). Atomic absorption studies revealed that the iron concentration in the soluble cellular fraction was maximal at 35 h of incubation and accounted for 1.3% of the total iron. However, at stationary phase of growth, most of the iron was deposited as a PE containing residue. A transmission electron microscope, equipped with an electron energy loss spectrometer (EELS), allowed the visualization of iron bodies within the bacterial cells. However, such metal inclusions were absent in cells isolated at stationary phase of growth.

Aluminum Elicits Exocellular Phosphatidylethanolamine Production in Pseudomonas fluorescens

Pseudomonas fluorescens ATCC 13525 was found to grow in a minimal mineral medium supplemented with millimolar amounts of aluminum, a known environmental toxicant. During the stationary phase of growth, the trivalent metal was localized in a phosphatidylethanolamine (PE)-containing residue. The concentration of PE in pellets ranged from 1.7 to 13.9 mg ml of culture(sup-1) in media supplemented with 1 to 30 mM aluminum. Although the gelatinous residue was observed during the stationary phase of growth, ultracentrifugation and dialysis experiments revealed that PE was produced from earlier stages of incubation and was associated with aluminum. A sharp diminution in the levels of PE and aluminum in the spent fluid was concomitant with the formation of the insoluble deposit. The aluminum content of the soluble cellular fraction increased during growth and reached an optimum of 1.85 mM of test metal at 45 h in cultures with 15 mM aluminum. Further incubation, however, led to a marked decrease in the cellular aluminum content, and during the stationary phase of growth, only trace amounts of the trivalent metal were detected in this fraction. When 45-h cells were incubated in fresh citrate medium, most of the intracellular aluminum was secreted in the spent fluid and citrate was rapidly consumed. Aluminum efflux was also observed in cultures in which d-glucose was substituted for citrate. However, no efflux of this trivalent metal was evident in media devoid of either citrate or d-glucose. Scanning electron microscopic studies and X-ray energy-dispersive analyses of the dialyzed supernatant aided in the visualization of nodule-like aluminum- and phosphorus-rich bodies associated with thread-like carbon-, oxygen-, and phosphorus-containing structures. Transmission electron microscopic and electron energy loss spectroscopic analyses revealed the presence of aluminum within bacteria after 45 h of incubation. Cells harvested after aluminum insolubilization did not shown aluminum inclusions. This aluminum-tolerant microbe may have potential application in bioremediation processes.

Purification and characterization of the cytoplasmic histone acetyltransferase B of maize embryos

From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B-type enzyme. Using 8 chromatographic steps, we achieved a 6700-fold purification of an enzymatically active protein with a molecular weight of ∼ 90 kDa. Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS-PAGE, suggesting that the native enzyme is a heterodimer. The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity. It was specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro. Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination. We established an electrophoretic system under non-denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS-PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations.

Influence of phosphate on aluminum tolerance inPseudomonas fluorescens

When grown in a minimal mineral medium containing 0.64 mM phosphate, Pseudomonas fluorescens attained a cellular yield comparable to that observed in a similar medium with 64 mM phosphate. However, the inclusion of aluminum in these two media evoked disparate responses to aluminium stress. In the latter culture, aluminum was immobilized as a gelatinous lipid-rich residue (Appanna et al., FEMS Microbiol. (1994) 119, 295–302), while in the former medium the trivalent metal was sequestered in soluble metabolite(s) localized in the spent fluid. In the medium with 0.64 mM phosphate, the cellular yield decreased by 10–40% in the presence of 3–15 mM aluminum. Citrate, the sole source of carbon to which the metal was complexed, was completely utilized and no significant change in exocellular protein or carbohydrate was evident. While only a trace amount of aluminum was recorded in the soluble cellular fraction, only 11% of the test metal was immobilized as a lipid residue. However, most of the metal was sequestered in metabolite(s) localized in the spent fluid. It appears that mechanism(s) initiated by the microbe to achieve aluminum homeostasis is dependent on the phosphate concentration in the medium.

Secretion in AtT-20 cells stably transfected with soluble synaptotagmins.

Synaptotagmin (p65) is an integral membrane secretory vesicle-specific protein with two cytoplasmic repeats homologous to the C2 regulatory domain of protein kinase C. Synaptotagmin has been implicated in the regulation of neurotransmitter release from nerve growth factor-differentiated PC12 cells and from synapses in Drosophila, Caenorhabditis elegans, and squid. To address the function of synaptotagmin in endocrine cells, fragments of rat synaptotagmin I were stably expressed in the mouse anterior pituitary cell line AtT-20. The logic of these experiments is that the fragments may interfere with the endogenous synaptotagmin machinery, thus producing a dominant-negative phenotype. Transfected cells expressed the expected fragments that were comprised of either the first C2 repeat, the second C2 repeat, or the entire cytoplasmic domain. The fragments were localized to both soluble and membrane-associated cellular fractions, despite the absence of the transmembrane domain. The second C2 repeat was shown to coimmunoprecipitate with endogenous synaptotagmin, suggesting that protein-protein interactions are mediating the membrane association of the fragments. These fragments had no effect on the targeting of regulated secretory vesicles or on regulated secretion as assayed by the release of ACTH and [3H]choline. Constitutive secretion assayed by the release of glycosaminoglycan side chains was also unaffected, as was the endocytic pathway monitored by the uptake and clearance of transferrin. These data suggest either the existence of a redundant pathway in secretion or that regulated membrane traffic in endocrine cells does not require synaptotagmin.

Production of enzymatically active rat protein disulfide isomerase in Escherichia coli.

We report the development of a bacterial expression system allowing high-level synthesis of enzymatically active rat protein disulfide isomerase (rPDI). After expression of the rpdi gene under control of the inducible trc promoter (Ptrc), a significant amount of soluble, active rPDI was detected in the periplasmic contents, which were released from the cells by cold osmotic shock. However, the exported molecules were incompletely or improperly processed, while the major amount of synthesized rPDI was in fact detected in the soluble cellular fraction. Substitution of the autologous eukaryotic export signal with the nucleotide (nt) sequence encoding the signal peptide (sOmpA) of the bacterial outer membrane protein A, and expression of the sompA::rpdi fusion gene under control of both the lpp promoter (Plpp) and the lac promoter-operator (POlac), resulted in high-level production of rPDI. Furthermore, the latter was efficiently exported into the periplasmic compartment, from where it was recovered as a soluble, fully active form with the sOmpA precisely removed. The synthesis of a small 21-kDa peptide accompanying the production of rPDI was also observed. This rPDI-related peptide (rPDIf), which represented a C-terminal fragment of rPDI including the second active site, arose by internal translation initiation within rpdi. Replacement of the presumed internal start codon by CTC completely eliminated the aforementioned phenomenon and resulted in the production of a slightly mutated, enzymatically active enzyme (rPDIm).

Analysis of Hepatitis C Virus Capsid, E1, and E2/NS1 Proteins Expressed in Insect Cells

The baculovirus/insect cell expression system was used to express the capsid protein and glycoproteins (e1 and e2/NS1) of the hepatitis C virus (HCV). Each polypeptide domain was expressed individually using two different constructs varying at their carboxy termini in order to retain or delete hydrophobic domains that may be involved in membrane association. The capsid proteins were transported to the nucleus where they formed a single large crystal-like inclusion. The capsid proteins were phosphorylated in insect cells. The e1 and e2 polypeptides were present in both the soluble and insoluble cellular fractions. Deletion of a hydrophobic domain in the carboxy terminus of e2 resulted in the polypeptide becoming soluble but not secreted. Deletion of the carboxy terminus of e1 had no effect on solubility. Both e1 and e2 were glycosylated, with variable glycosylation of e1 giving rise to a series of polypeptides varying in apparent molecular weight Co-infection of insect cells with viruses expressing e1 and e2 resulted in a complex that permitted the coimmunoprecipitation of e1 with antibodies to e2 end vice versa. Immunofluorescence staining of insect cells expressing e1 and e2 indicated that reactivity to e2 was more prevalent in anti-HCV positive human sera.

Protein differences in tau mutant hamsters: candidate clock proteins

In the tau mutant hamster, the period of the circadian rhythm is shortened from about 24 h to about 22 h in heterozygotes and to about 2 h in homozygotes. Understanding the biochemical basis of the period changes in the tau mutant may elucidate the regulation of the vertebrate pacemaker. Using two-dimensional gel electrophoresis, we have found two sets of proteins that differ between the different genotypes. P33 tau (about 33 kDa; pI 6.5) was found in all gels from wild type and heterozygous animals, but was absent in gels from all except one of the homozygous mutant animals. P32 tau (about 32 kDa; pI 4.8) was a chain of spots, which showed a striking difference in pattern between gels from wild type animals and from mutant animals. P33 tau was greatly enriched in soluble cellular fractions, whereas P32 tau was found only in insoluble fractions. These differences between P33 tau and P32 tau were apparent in gels from both SCN and cortical tissue, suggesting that both proteins are distributed throughout the brain. These proteins should be useful as new tools to explore the biochemistry of circadian pacemakers.

Determination of alpha B crystallin aggregation: a new alternative method to assess ischemic damage of the heart.

alpha B crystallin, a heat-shock-like protein, is a major component of the soluble protein fraction of the heart and is thought to play a protective role in stress situations. During an ischemic episode, the cytosol of cardiomyocytes acidifies, thus causing the aggregation of the protein with cytoskeletal elements. After homogenization of the tissue, alpha B crystallin can then be recovered with the insoluble cell components. This study investigated the change of the solubility properties of crystallin in the ischemic heart. The distribution of crystallin in the soluble and insoluble cellular fractions was determined by centrifugation of heart homogenates and immunoblot analysis with anti-alpha B crystallin antibodies. The proportion of aggregated alpha B-crystallin increased in hearts reperfused after total normothermic ischemia of increasing severity. alpha B crystallin aggregation was proportional to the amount of lactate dehydrogenase activity released by the hearts and was inversely correlated to the ability of the hearts to recover contractile activity after the ischemic episode. This study shows that the amount of aggregated crystallin can be used as a new marker for the ischemic damage of the heart. Biopsies of a few milligrams are sufficient for the analysis.

Characterization of a calcium and diacylglycerol-activated and phospholipid-dependent protein kinase in the pupal brain of the tobacco hornworm, Manduca sexta: The particulate associated enzyme

A particulate associated, calcium and diacylglycerol-activated, phospholipid-dependent protein kinase (protein kinase C; PKC) has been demonstrated in the pupal brain of the tobacco hornworm, Manduca sexta. The particulate enzyme could be measured accurately only after its solubilization by non-ionic detergents, Triton X-100 being the most effective. The basic enzymatic properties of the particulate associated PKC are similar to those of the cytosolic brain enzyme. Maximal enzyme activity was obtained in the presence of 0.75 mM Ca 2+ and 200 μg/ml phosphatidylserine. Diacylglycerol (1,2 diolein; 50 μg/ml) further enhanced both PKC activity and Ca 2+ sensitivity in the presence of an optimal concentration of phosphatidylserine. Particulate PKC is inhibited by trifluoperazine in a dose-dependent manner with an IC 50 of approx. 30 μM. Subcellular distribution studies revealed that 86% of the pupal brain PKC activity is present in the soluble cellular fraction. A study of PKC activity during pupal-adult development revealed that the specific activity of day 9 pharate adult brain PKC was 6-fold higher than that observed for day 0 pupal brains. SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated that a microsomal enriched 34 kDa peptide was phosphorylated by PKC in the presence of Ca 2+ and phosphatidylserine.

Purification and characterization of the DNA-binding protein Ner of bacteriophage Mu

The construction is described of a plasmid (pL- ner) which directs the high-level production of the bacteriophage Mu Ner protein in Escherichia coli. The protein, recovered m the soluble cellular fraction, was susceptible to in vivo proteolytic processing, in many host strains, but not in E. coli B, a natural lon − prototroph. A simple purification method is described which takes advantage of the basic nature of the protein. The purified protein was shown to be physically and chemically homogeneous and to have an amino acid sequence identical to that predicted for the authentic protein. The protein was also shown to have in vitro biological activity, as measured by specific binding to a DNA fragment containing the consensus Ner-binding sequence, and in vivo biological activity as the protein produced by the pL- ner plasmid allowed lysogenic-like maintenance of a Mu prophage c mutant unable to synthesise a functional Mu repressor.

Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens.

Using an N-bromosuccinimide cleavage fragment of histone H1 as a relatively specific substrate for protein kinase C, we evaluated the partitioning of this kinase activity between soluble and particulate cellular fractions in 3T3-L1 fibroblasts. In confluent, serum-deprived cells, protein kinase C activity was approximately equally divided between soluble and detergent-extractable particulate fractions; both rapidly growing and transformed cells appeared to contain higher levels of particulate enzyme activity. Soluble protein kinase C activity and immunoreactivity decreased to virtually undetectable levels after exposure of the cells to phorbol 12-myristate 13-acetate (PMA), associated with a commensurate increase in particulate kinase activity and immunoreactivity. In intact cells, PMA appeared to cause a shift of immunoreactive protein kinase C from the cytosol to the perinuclear region, as assessed by immunofluorescent microscopy; however; subcellular fractionation revealed that PMA caused increases in the protein kinase C activity associated primarily with non-nuclear membranes. Exposure of the cells to sn-1,2-dioctanoylglycerol resulted in a modest and transient membrane association of protein kinase C, whereas platelet-derived growth factor, fibroblast growth factor, and bombesin caused no detectable increases in the membrane association of the kinase. Activation of protein kinase C by growth factors in fibroblasts may occur without the gross disturbances in intracellular kinase location which occur in response to phorbol esters.

Transglutaminase (TG) involvement in early embryogenesis

Transglutaminase (TG) has been examined in different stages of preimplantation mouse embryogenesis. The specific activity of this enzyme in the soluble cellular fraction increases 2-fold from 2-cell embryos to 8-cell morulae and 4-fold from 2-cell embryos to blastocyst. The same developmental profile was seen when either N,N′-dimethylcasein or endogenous substrates were used in the TG assay. Using high-speed supernatants from different stage embryos as a source of enzyme and [ 3H]putrescine as acyl acceptor, the major acyl donor components were tubulin and a high molecular weight (HMW) cross-linkage product, as assessed by electrophoresis and immunoblotting. When either assembled or monomeric cytoskeleton proteins were compared as substrates, microtubules were the best acyl donors. These studies indicate that TG activity is modulated during the changing demands of blastomeres for microtubule cytoskeleton in early embryogenesis.

RNA synthesis and aminoacyl transfer RNA activity of L-2071 cells exposed to dieldrin and DDT

In L-2071 tissue culture cells, 50 ppm (50 Μg/ml culture medium) each of dieldrin or DDT reduced total cell count and the rate of14C-leucine incorporation into protein and14C-uridine incorporation into total RNAs; this was the result of an increased rate of14C-uridine incorporation into cytoplasmic soluble and 4S RNA cellular fraction. Total amino acid acceptor activity decreased with dieldrin and DDT treatments, which suggests an actual decrease in functional tRNAs. Dieldrin reduced glutamyl- and aspartyl-tRNA activities whereas phenylalanyl-, tyrosyl-, threonyl-, leucyl-, isoleucyl-, seryl-, and prolyl-tRNA activities decreased with both pesticides. Valyl-, lysyl-, glycyl-, alanyl-, histidyl-, and arginyl-tRNA activities were only slightly changed. The effect of dieldrin was generally greater than that of DDT. In addition, there was degradation of 28S and 18S RNAs to fragments isolated with 4S RNAs. Cells treated with 0.5 and 10 ppm levels of dieldrin demonstrated decreased14C-uridine incorporation into 28S RNAs and increased incorporation into 18S and 4S RNAs after 3 hours and 3 to 6 hours, respectively. Incorporation levels returned to near control levels, thereafter, in whole cell preparations, but remained different from controls when nuclear and cytoplasmic fractions were assessed individually.

On the degradation of intracellular RNA by ribonucleases

1. 1. The kinetic and the specificity of two RNases purified fom the insect. C. capitata have been studied. 2. 2. These two enzymes exhibit preference to degrade large polynucleotides. 3. 3. The alkaline enzyme is located in the soluble cellular fraction and the acid enzyme is also associated to microsomes and lysosomes. 4. 4. Ahypothesis about the physiological role of these two insect enzymes in the degradation of the intracellular RNA is proposed.

Transport and phosphorylation of xylitol by a fructose phosphotransferase system in Streptococcus mutans.

The purpose of this work was to explain how the caries-preventive agent xylitol interferes with the growth of Streptococcus mutans. It was found that the xylitol-sensitive strain of S. mutans 27352 (serotype g) and LG1 (serotype c) took up 14C-xylitol when the labelled pentitol was added to cells growing at the expense of glucose. Uptake of xylitol by growing cells of S. mutans 27352 XR and LG1 XR, two xylitol-insensitive spontaneous mutants, and of S. mutans GS5-2, which was also insensitive to xylitol, was practically inexistent under the same conditions. Alkaline phosphatase treatment followed by enzymatic analysis and thin-layer chromatography revealed that the accumulated product was xylitol phosphate. Intracellular concentrations of 5–7 mM for resting cells and of up to 60 mM for growing cells were calculated. Xylitol was phosphorylated at the expense of phosphoenolpyruvate by toluenized cells of S. mutans LG1, but not by toluenized cells of GS5–2 and S. mutans LG1 XR. The phosphorylation of xylitol was dependent on phosphoenolpyruvate and required the presence of both soluble and membrane cellular fractions in the reaction mixture. This indicated that xylitol was transported and phosphorylated by a phosphoenolpyruvate: sugar phosphotransferase system. The phosphoenolpyruvate-dependent phosphorylation by isolated membranes of S. mutans LG1 in the presence of the soluble fraction was inhibited by fructose but not by glucose, mannose or galactose. Measurement of phosphoenolpyruvate: phosphotransferase activities in isolated membrane revealed that strain 27352 and LG1 had activities for fructose and xylitol; membrane from 27352 XR and LG1 XR had very little activity for fructose and xylitol. It was concluded that xylitol was transported and phosphorylated by a constitutive phosphoenolpyruvate:fructose phosphotransferase system in S. mutans. The data suggested that xylitol toxicity in S. mutans is caused by the intracellular accumulation of xylitol phosphate.