Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.     

Tyrosine protein phosphorylation in murine B lymphocytes by stimulation with lipopolysaccharide from Porphyromonas gingivalis

Abstract The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements. Three different insertion sequence (IS) elements entrapped in sacB were isolated. The IS elements IS- Bl and IS- Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum , respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp. Their inverted repeats showed homology. In contrast, the IS element IS- Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication. IS- Cg and IS- Rf were not restricted to their original host strains, and we also found strains harbouring more than one element.     

Tyrosine phosphorylation of the Tio oncoprotein is essential for transformation of primary human T cells

Human T cells are transformed to antigen-independent permanent growth in vitro upon infection with herpesvirus saimiri subgroup C strains. The viral oncoproteins required for this process, StpC and Tip, could be replaced by Tio, the oncoprotein of herpesvirus ateles. Here we demonstrate that proliferation of lymphocytes transformed with Tio-recombinant herpesvirus saimiri required the activity of Src family kinases. Src kinases had previously been identified as interaction partners of Tio. This interaction was now shown to be independent of any of the four tyrosine residues of Tio but to be dependent on an SH3-binding motif. Mutations within this motif abrogated the transforming capabilities of Tio-recombinant herpesvirus saimiri. Furthermore, kinase interaction resulted in the phosphorylation of Tio on a single tyrosine residue at position 136. Mutation of this residue in the viral context revealed that this phosphorylation site, but none of the other tyrosine residues, was required for T-cell transformation. These data indicate that the interaction of Tio with a Src kinase is essential for both the initiation and the maintenance of T-cell transformation by recombinant herpesvirus saimiri. The requirement for the tyrosine phosphorylation site at position 136 suggests a role for Tio beyond simple deregulation of the kinase.     

Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes

The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood. We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC). In addition, we related these early events to later events in B-cell activation, including cell proliferation. Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells. Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation. Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events. Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and [3H]TdR incorporation induced by all stimuli. We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins. These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.     

Tyrosine phosphorylation of interleukin 2 receptor-related proteins in phytohemagglutinin-activated human lymphocytes

Three classes of proteins (mol wts 70k, 64k and 45k) having the characteristics of interleukin 2 receptor were detected in phytohemagglutinin-activated human lymphocytes using two monoclonal antibodies which recognize distinct epitopes on the receptor. It was shown that at least portions of these proteins were phosphorylated on tyrosine by analyses for phosphotyrosine by immunoblotting and by immunoaffinity chromatography with antibodies to phosphotyrosine. In addition an iodinated phosphotyrosine derivative was identified in partial hydrolysates of these proteins iodinated in vitro.     

Phosphorylation of HsMis13 by Aurora B kinase is essential for assembly of functional kinetochore

Chromosome movements in mitosis are orchestrated by dynamic interactions between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that phosphorylation of human HsMis13 by Aurora B kinase is required for functional kinetochore assembly in HeLa cells. Aurora B interacts with HsMis13 in vitro and in vivo. HsMis13 is a cognate substrate of Aurora B, and the phosphorylation sites were mapped to Ser-100 and Ser-109. Suppression of Aurora B kinase by either small interfering RNA or chemical inhibitors abrogates the localization of HsMis13 but not HsMis12 to the kinetochore. In addition, non-phosphorylatable but not wild type and phospho-mimicking HsMis13 failed to localize to the kinetochore, demonstrating the requirement of phosphorylation by Aurora B for the assembly of HsMis13 to kinetochore. In fact, localization of HsMis13 to the kinetochore is spatiotemporally regulated by Aurora B kinase, which is essential for recruiting outer kinetochore components such as Ndc80 components and CENP-E for functional kinetochore assembly. Importantly, phospho-mimicking mutant HsMis13 restores the assembly of CENP-E to the kinetochore, and tension developed across the sister kinetochores in Aurora B-inhibited cells. Thus, we reason that HsMis13 phosphorylation by Aurora B is required for organizing a stable bi-oriented microtubule kinetochore attachment that is essential for faithful chromosome segregation in mitosis.     

Tyrosine phosphorylation events during coxsackievirus B3 replication.

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.     

T lymphocyte aggregation with immobilized anti-TCR-antibodies is dependent upon energy and microfilament assembly

An assay has been developed to quantitate the binding of beads coated with anti-T cell receptor (TCR) monoclonal antibodies (MoAb) to T lymphocytes. The Ab used were a hamster MoAb, 145.2C11 (2C11), directed against the epsilon chain of the CD3 complex of the murine TCR, and a murine MoAb, F23.1, directed against the V beta 8-encoded determinant of the alpha/beta heterodimer of the TCR. Ab were adsorbed onto polystyrene beads and the beads labeled with [125I]bovine serum albumin [( 125I]BSA). The labeled, Ab-coated beads were mixed at 4 degrees C with murine, cloned T-helper (Th) cells and contact between beads and cells was promoted by centrifugation. The mixtures were incubated at 37 degrees C for 10-20 min, and unbound beads were separated from cell-bound beads by Percoll gradient centrifugation. Beads coated with anti-TCR Ab formed stable conjugates with Th cells; an average of 6-10 2C11 Ab-coated beads/cell, or 10-15 F23.1 Ab-coated beads/cell was measured under optimal conditions. Beads coated with control Ab (hamster or mouse IgG) did not appreciably bind to the cells. Conjugation with 2C11 Ab-coated beads could be prevented by coating the cells with soluble 2C11 Ab, but not with soluble F23.1 Ab. Blocking the CD3 epsilon chain with soluble 2C11 Ab also reduced conjugate formation with F23.1 Ab-coated beads, suggesting a steric hindrance phenomenon. The extent of conjugation depended on the density of immobilized Ab. Maximum conjugation was observed when 100 micrograms of 2C11 Ab was used to coat 10(6) beads; higher Ab amounts did not further increase binding. Increasing the bead to cell ratio in the mixture increased binding, reaching optimal binding at 300:1, irrespectively of the amount of Ab adsorbed onto the beads. Stable binding of anti-TCR Ab-coated beads to T cells was temperature and energy dependent. It was prevented when glucose was removed from the medium and the glycolysis inhibitor, 2-deoxy-D-glucose was added, or when cells were treated with sodium azide. Conjugate formation was prevented by pretreatment of the cells with cytochalasins, indicating that microfilament assembly was essential. Microtubules were not involved, as vinca alkaloids were without effect. This novel assay system provides a simple means of studying aspects of TCR function including its physical and metabolic regulation.     

Tyrosine phosphorylation is an obligatory event in IL-2 secretion.

Activation of T lymphocytes leads to the production of the T cell growth factor IL-2 that regulates T cell proliferation. This activation is associated with several potential intracellular signalling events including increased activity of phospholipase C (PLC) and resultant increases in production of inositol phosphates and diacylglycerols. In addition, phosphorylation of specific intracellular proteins on serine, threonine, and tyrosine residues increases. The role of each of these events in IL-2 production is unclear. Using Western blotting with antiphosphotyrosine antibodies, we demonstrate that activation of murine T cells with mitogenic lectins or anti-CD3 antibodies leads to a rapid increase in tyrosine phosphorylation of proteins of 120, 72, 62, 55, and 40 kDa. Similar patterns of antiphosphotyrosine antibodies reactivity were observed in splenocytes, a T cell hybridoma, and a T lymphoma. Tyrosine phosphorylation was detectable within minutes of addition of mitogenic lectins and persisted for at least 6 h. Pretreatment of the cells with pertussis toxin did not inhibit tyrosine phosphorylation indicating that a pertussis toxin-sensitive G protein is not involved in signal transduction. Neither increasing cytosolic-free calcium nor activating protein kinase C mimicked the effects of mitogenic lectins suggesting that tyrosine phosphorylation was not a consequence of activation of PLC. This was confirmed by demonstrating that mitogenic lectins induced similar patterns of tyrosine phosphorylation in cells in which activation of the TCR leads to increased PLC activity and in cells in which PLC is not stimulated. To test whether tyrosine phosphorylation is linked to IL-2 secretion, we determined the effect of three specific tyrosine kinase inhibitors (tyrphostins) on tyrosine phosphorylation, IL-2 secretion, and cellular proliferation. The concentration dependence of inhibition of tyrosine phosphorylation and IL-2 production were similar. However, higher concentrations of the tyrphostins were required to inhibit constitutive proliferation of the T cell line indicating that inhibition of IL-2 secretion was not secondary to nonspecific toxic effects of the tyrphostins. Addition of the tyrphostins after mitogenic lectin decreased the amount of tyrosine phosphorylation and IL-2 secretion in parallel. This indicates that both tyrosine kinases and phosphatases are activated and that continuous tyrosine phosphorylation is likely required for IL-2 secretion. Therefore, tyrosine phosphorylation appears to represent an obligatory event in the transmembrane signaling processes that lead to IL-2 secretion.     

Tyrosine phosphorylation in human lymphomas

In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated whether oncogenic tyrosine kinase activation also occurs in other categories of lymphoma by staining 145 cases of lymphoma covering those tumours with a range of different subtypes including those with morphological similarity to ALK-positive anaplastic large cell lymphoma (ALCL). Twelve cases of the borderline malignant disorder lymphomatoid papulosis were also studied. Twenty seven of the 28 cases of ALK-positive ALCL showed the extensive cytoplasmic labelling for phosphotyrosine in the neoplastic cells. The remaining case containing moesin-ALK exhibited membrane-associated phosphotyrosine expression. There was no nuclear phosphotyrosine labelling in any of the ALK-positive ALCL, even though ALK was present within the cell nuclei in 23 of the tumours. Variable degrees of phosphotyrosine labelling, usually membrane-restricted, were observed in 7/40 cases of ALK-negative ALCL, 9/29 cases of diffuse large B-cell lymphoma, 3/6 cases of mediastinal B-cell lymphoma, 2/7 cases of Hodgkin's lymphoma, 3/6 cases of peripheral T-cell lymphomas unspecified, 4/6 cases of B-cell chronic lymphocytic leukaemia, 2/6 cases of follicular lymphomas and 2/12 cases of lymphomatoid papulosis studied. However none of these phosphotyrosine-positive cases showed the strong cytoplasmic labelling comparable to that seen in ALK-positive lymphoma. We conclude that activation of a tyrosine kinase is probably not a major oncogenic event in lymphomas other than ALK-positive ALCL.     

Tyrosine phosphorylation in human neutrophil

Protein tyrosine phosphorylation in human neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. The addition of the human hormone granulocyte-macrophage colony stimulating factor to human neutrophils caused an increase in the tyrosine phosphorylation levels of several proteins. The increases in at least two of these proteins having molecular masses of 40 kDa (p40) and 54 kDa (p54) were rapid and were inhibited in pertussis toxin treated cells. The newly synthesized tyrosine kinase inhibitor ST 638 inhibited the increases in the levels of the tyrosine phosphorylation in p92, p78, p54 and p40 proteins. The epidermal growth factor receptor tyrosine kinase inhibitors were less effective. The addition of the chemotactic factor fMet-Leu-Phe to human neutrophils also caused an increase in tyrosine phosphorylation in some of these proteins. The pattern of the fMet-Leu-Phe-induced tyrosine phosphorylation was different from that produced by GM-CSF. The increases were also inhibited by ST 638. In addition, ST 638 inhibited superoxide production but not actin polymerization in control and GM-CSF-treated cells stimulated with fMet-Leu-Phe. Moreover, the active but not inactive phorbol esters increase the tyrosine phosphorylation only in the 40 kDa protein. These results suggest several points: (a) some of the responses produced by GM-CSF and fMet-Leu-Phe are mediated through tyrosine phosphorylation, (b) the GM-CSF receptor is coupled to a pertussis toxin sensitive G-protein, (c) the 40 kDa protein is probably the Gi alpha 2, and (d) the 78 or the 92 kDa protein is most likely the receptor for GM-CSF, which indicates that the receptor may have a tyrosine kinase domain.     

Oxidant-dependent phosphorylation of p40phox in B lymphocytes

As with the neutrophil NADPH oxidase, the B lymphocyte NADPH oxidase consists of a membrane-bound flavocytochrome b and regulatory factors including Rac and the cytosolic phox protein triad p67phox, p47phox, and p40phox. Here we demonstrate by phosphoamino acid analysis and the use of the potent PKC inhibitor GFX that, in response to stimulation of B lymphocytes with sodium orthovanadate and H(2)O(2), the p40phox component of the cytosolic phox triad is selectively phosphorylated on serine and threonine residues by a PKC-type protein kinase. The pattern of p40phox phosphorylation was closely related to the kinetics of tyrosine phosphorylation of PKC-delta, the main PKC isotype of B lymphocytes. Blocking H(2)O(2)-dependent tyrosine phosphorylation of PKC by genistein resulted in inhibition of p40phox phosphorylation. The correlation between the tyrosine phosphorylation of PKC-delta and the serine/threonine phosphorylation of p40phox, together with the inhibition of p40phox phosphorylation by rottlerin, a selective inhibitor of PKC-delta, makes the activated PKC-delta a likely candidate in the process of the oxidant-dependent phosphorylation of p40phox in B cells.     

Tyrosine phosphorylation of the Janus kinase 2 activation loop is essential for a high-activity catalytic state but dispensable for a basal catalytic state

The phosphorylation of an "activation loop" within protein kinases is commonly associated with establishing catalytic competence, and phosphorylation of the Tyr(1007) residue in the activation loop of Janus kinase 2 (JAK2) has been shown to be essential for intracellular propagation of cytokine-initiated signaling. We provide evidence for the presence of a basal activity state of JAK2, which was observed in the absence of activation loop phosphorylation. Phosphorylation of the JAK2 activation loop was essential for conversion to the high-activity state, characterized by high-efficiency ATP utilization during autophosphorylation. Mutagenesis of activation loop tyrosine residues Tyr(1007/1008) to phenylalanine residues impaired, but did not abolish, the enzyme's ability to autophosphorylate. The activation loop mutant JAK2 could also transphosphorylate an inactive JAK2 fragment coexpressed in Sf21 cells, providing evidence of exogenous substrate phosphorylation. The mutant enzyme remained in a basal activity state characterized by low-efficiency ATP utilization during autophosphorylation. Mutagenesis of a critical Lys(882) residue to a glutamate residue abolished all evidence of kinase activity, confirming that the observed activity of Tyr-to-Phe mutants was not due to another kinase. Our data are consistent with the proposal that JAK2 is an inefficient but active enzyme in the absence of activation loop phosphorylation and is capable of conversion to a high-activity state by autophosphorylation under physiological ATP concentrations. This theoretically precludes the need for an upstream activating kinase. The activation process of JAK2 may be envisioned as a multistate process involving at least two kinetically distinct states of activity.     

Tyrosine phosphorylation of CD19 in pre-B and mature B cells.

Cross-linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen-specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg-associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross-linking sIg results in an increase in Tyr phosphorylation of the lineage-restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr-phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg- pre-B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross-linking, Tyr phosphorylation of CD19 was induced in sIg- pre-B cell lines. CD19 cross-linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre-B cell development.     

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.     

Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin.

Activation of both T and B lymphocytes through their membrane receptors for antigen is known to induce breakdown of inositol phospholipids. In addition, T cell activation by antigen is accompanied by increased protein tyrosine phosphorylation of components of the T cell antigen receptor. We now provide evidence that B cell activation through membrane immunoglobulin is also coupled to stimulation of protein tyrosine kinase activity. One potential candidate for a B lymphocyte protein tyrosine kinase is an 80 kd molecule that is itself phosphorylated at tyrosine residues in response to stimulation with anti-immunoglobulin antibodies.     

Filamin-regulated F-actin assembly is essential for morphogenesis and controls phototaxis in Dictyostelium

Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. Deletion of individual rod repeats also changed their subcellular localization, and mutant filamins in general were less enriched at the cell cortex as compared with the full-length protein and were increasingly present in the cytoplasm. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extracellular-matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.     

Tyrosine83 is essential for the activity of E. coli galactoside transacetylase

The gene (lacA) coding for Escherichia coli galactoside transacetylase was cloned into the pTrcHisB plasmid, and the corresponding hexahistidine-tagged enzyme was over-expressed and purified. The kinetic constants of the tagged protein were determined, yielding values in excellent agreement with previous observations reported for the natural enzyme. LacA Tyrosine83 was then substituted with a Valine: by comparing the K(m) and k(cat) values observed for wild type and mutant enzymes using isopropyl-thio-beta-d-galactopyranoside or p-nitrophenyl-beta-d-galactopyranoside as substrates, Tyrosine83 was identified as an essential residue for the catalytic activity of E. coli galactoside transacetylase.