Inhibition of Lysozyme by Some Copolymers of Amino Acids<sup>*</sup>

Sela, Michael; Steiner, Lisa A.

doi:10.1021/bi00903a004

Cited by 47 publications

(11 citation statements)

References 24 publications

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“…Such substrate inhibition presumably is due to the presence of a fraction of the tyrosine residues in the random copolymer that are not efficient phosphoacceptor sites yet can still be bound by the enzyme. Poly(Glu80Tyr20) at high concentrations is known to inhibit a variety of other enzymes (44,45), including the protein-tyrosine kinase pl3Ov-fPs (4 Phosphorylation of p40 on tyrosine in vitro and in vivo. A preparation of p40, purified as described above and then concentrated by adsorption to and elution from Mono S FLPC (Fig.…”

Section: Methodsmentioning

confidence: 99%

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Dailey¹,

Schieven²,

Lim³

et al. 1990

Mol. Cell. Biol.

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Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G. S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinitypurified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPKI) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.Protein-tyrosine kinases are involved in growth control and malignant transformation of animal cells (22,23). A large number of protein-tyrosine kinases, including a number of growth factor receptors (48), have been described in both vertebrates and invertebrates (20). The molecular mechanisms by which these enzymes affect cellular growth and induce transformation remain'unclear, despite the identification of some substrates for these enzymes (11,14,22,38). To further elucidate the role of protein-tyrosine kinases in normal cells, we began an examination of the proteintyrosine kinases of bakers' yeast (Saccharomyces cerevisiae). S. cerevisiae offers an attractive system for study of eucaryotic growth control because of its short generation time, its small genome size, the availability of numerous cell division cycle mutations (37), and the ease of application of molecular genetic methods (such as gene replacement) (2).We have demonstrated previously that S. cerevisiae contains a soluble activity capable of phosphorylating both exogenous substrates, such as poly (Glu80Tyr20) product of the cdc2+ gene, p34cdc2, is phosphoryl...

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Section: Methodsmentioning

confidence: 99%

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Dailey¹,

Schieven²,

Lim³

et al. 1990

Mol. Cell. Biol.

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“…Some basic proteins, such as lysozyme, are inhibited in a nonspecific way by a wide range of acidic substances, e.g. nucleic acids, acidic polysaccharides and acidic proteins (Salton, 1957;Cattan & Bourgoin, 1968;Caputo, 1955;Sela & Steiner, 1963;Pryme, Joner & Jensen, 1969). Samples of purified glucosaminidase (100 units/rnl) were incubated with 0.4mg and 4mg of RNA, DNA, hyaluronic acid or teichoic acid for 15min at 200C, and for 24h at 40C.…”

Section: Resultsmentioning

confidence: 99%

Bacteriolytic enzymes from Staphylococcus aureus. Properties of the endo-β-N-acetylglucosaminidase

Wadström

1970

Biochemical Journal

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An extracellular bacteriolytic endo-beta-N-acetylglucosaminidase has been purified and its specificity of action has been investigated (Wadström & Hisatsune, 1970a,b). Some enzymic properties, such as optimum pH for enzyme activity on whole cells and cell walls of Micrococcus lysodeikticus and Staphylococcus aureus and optimum pH for stability, have been studied. The activity was maximum in 0.05m-tris-hydrochloric acid buffer, pH7.0. A higher ionic strength inhibited cell-wall hydrolysis. Since the crude and purified enzymes were found to be unstable on storage, the stabilizing and inhibiting effects of several compounds were investigated. Several heavy metal ions inactivated the enzyme at very low concentrations. Thiol compounds stabilized and thiol-reacting compounds partly inhibited the activity. Crude and purified glucosaminidase was found to be heat-stable at acidic pH and unstable at alkaline pH as previously found for several lysozymes (endo-beta-N-acetylmuramidases). Other properties of the staphylococcal enzyme and hen's-egg-white lysozyme have been compared, since the modes of action of the two are quite similar (Wadström & Hisatsune, 1970b).

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“…The digestion of ribonucleic acid by pancreatic ribonuclease was stopped completely, at pH 5.0, by relatively small amounts of the inhibitory copolymer. We tested these copolymers also for inhibition of lysozyme (20) and trypsin (21) with similar results.…”

Section: Ribonuclease and Other Enzymesmentioning

confidence: 92%

From Proteins and Protein Models to Their Use in Immunology and Immunotherapy

Sela

2003

Journal of Biological Chemistry

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Inhibition of Lysozyme by Some Copolymers of Amino Acids^*

Cited by 47 publications

References 24 publications

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Bacteriolytic enzymes from Staphylococcus aureus. Properties of the endo-β-N-acetylglucosaminidase

From Proteins and Protein Models to Their Use in Immunology and Immunotherapy

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Inhibition of Lysozyme by Some Copolymers of Amino Acids*

Cited by 47 publications

References 24 publications

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Bacteriolytic enzymes from Staphylococcus aureus. Properties of the endo-β-N-acetylglucosaminidase

From Proteins and Protein Models to Their Use in Immunology and Immunotherapy

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Inhibition of Lysozyme by Some Copolymers of Amino Acids^*