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L A Pinna

Publications and source records attributed to L A Pinna.

At least 127 records · Page 7Linked to original sources
PubMed1992

Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme.

Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce the loss of kinase activity, strongly suggesting a protective function for the beta subunit. Assaying different peptides for specificity toward the recombinant alpha subunit and the recombinant reconstituted enzyme, showed that the presence of the beta subunit could modify the specificity of the catalytic alpha subunit. Therefore, a dual function for the beta subunit is proposed which confers both specificity and stability to the catalytic alpha subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55-71 segment of the human beta subunit, counteracts the stimulatory effect of the beta subunit on the alpha subunit activity and partially substitutes the beta subunit in conferring thermal stability to the alpha subunit. No such effect is induced by the peptide MSSSEEVSW, reproducing the N-terminal segment of the beta subunit including the autophosphorylation site. It is suggested that the acidic domain of the beta subunit, encompassing residues 55-71, plays a role in the interactions between the beta and alpha subunits.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of maize seedling casein kinase IIB, a monomeric enzyme immunologically related to the alpha subunit of animal casein kinase-2.

Casein kinase IIB (CKIIB), a protein kinase related to animal casein kinase-2 (CK2), has been purified to homogeneity. It appears to be a monomeric enzyme, composed by an individual 39 kDa subunit, homologous to the alpha/alpha' subunits of animal CK2 and devoid of the autophosphorylatable 25-kDa alpha subunit of animal CK2, which display an heterotetrameric alpha 2 beta 2/alpha alpha' beta 2 structure. Such a conclusion is supported by the following lines of evidence: (1) CKIIB displays an apparent 39,000 Mr by gel filtration on Ultrogel AcA 34 and it gives rise to a single prominent protein band of similar Mr (38,000) upon SDS/PAGE; (2) upon incubation of the enzyme with [32P]ATP, no radiolabeled bands are detectable which might be attributable to either canonical or atypical beta subunits; (3) the 39-kDa band immunoreacts with antisera that recognize the alpha subunit of rat and chicken CK2; (4) conversely, no component immunologically related with the beta subunit could be detected in CKIIB by Western-blot analyses with antisera that recognize animal beta subunits; (5) the recombinant beta subunit of human CK2 is readily phosphorylated by CKIIB, the reaction being prevented, rather than stimulated, by polylysine, a behaviour typical of animal CK2 autophosphorylation. While the responsiveness of CKIIB to either heparin inhibition or polylysine stimulation are reminiscent of those of animal CK2, its peptide substrate specificity is significantly different and its thermolability is increased. Altogether these data would indicate that maize seedling CKIIB represents a naturally occurring monomeric form of CK2 devoid of non-catalytic subunits. Its properties, compared to those of animal CK2, suggest that the beta subunits of animal CK2 may be responsible for structural modifications conferring an altered specificity and an increased stability to the catalytic subunit.

Adenosine Triphosphate↗
PubMed1992

The comparative efficiencies of the Ser(P)-, Thr(P)- and Tyr(P)-residues as specificity determinants for casein kinase-1.

The beta-casein derived phosphopeptide, Glu-Glu-Ser(P)-Glu-Glu-Ser-Ile-Thr-NHMe and two derivatives in which the Ser(P)-residue is replaced by the Thr(P)- and Tyr(P)-residue have been compared for their susceptibility to phosphorylation by casein kinase-1. While both the Ser(P)- and Thr(P)-peptides are good substrates with similar kinetic constants, the Tyr(P)-peptide is a substrate as poor as the unphosphorylated derivative EEEEESIT, exhibiting a 21-fold higher Km and 6-fold lower Vmax values. While prior dephosphorylation of the Ser(P)-peptide caused a marked loss in its phosphoacceptor capacity, prior dephosphorylation of the Tyr(P)-peptide caused no significant change in its poor phosphoacceptor capacity. Thus the order of efficiency of phosphoaminoacids as specificity determinants for casein kinase-1 was found to be Ser(P)=Thr(P) much greater than Tyr(P) and this order is markedly different from Tyr(P) greater than Ser(P) much greater than Thr(P) which was previously established for casein kinase-2 [Meggio et al. (1991) FEBS Lett. 279, 307-309].

Amino Acid Sequence↗
PubMed1991

Substitution of phosphotyrosine for sulphotyrosine in biologically active peptides. Enzymatic phosphorylation of a progastrin peptide confers immunoreactivity reminiscent of the sulphated derivative.

The peptide SAEEEDQYN, corresponding to the carboxyl-terminal tryptic fragment of rat progastrin, whose penultimate tyrosyl residue is sulphated in the native peptide, is phosphorylated with Km values of 120 and 180 microM by two spleen tyrosine protein kinases, termed TPK-IIB and TPK-III, respectively. Another spleen tyrosine protein kinase related to the src family (TPK-I/lyn) is poorly active toward this peptide, displaying a Km 6.5 mM. The Tyr-phosphorylated peptide is recognized by an antibody (L304), which reacts with both sulphated and unmodified peptides, while it is not recognized by a second antibody (L303), which reacts with unmodified peptide yet not with the sulphated derivative. These data, in conjunction with previous observations (Hofsteenge, J., Stone, S.R., Donella-Deana, A. and Pinna, L.A. (1990) Eur. J. Biochem. 188, 55-59) support the view that phosphotyrosine is an effective surrogate for sulphotyrosine in a wide spectrum of biological activities.

Amino Acid Sequence↗
PubMed1991

Specificity determinants for tyrosine protein kinase. A study with recombinant hirudin mutants.

Hirudin, the powerful anticoagulant agent of leech (Hirudo medicinalis) saliva, was readily phosphorylated by two spleen tyrosine protein kinases (TPK-IIB and TPK-III) at Tyr63 with Km values of 238 microM and 74 microM, respectively. The synthetic tridecapeptide DGDFEEIPEEYLQ, corresponding to the hirudin 53-65 C-terminal fragment, was phosphorylated even more efficiently than hirudin itself. Four hirudin mutants, in which one or more of the glutamic acids at positions 57, 58, 61 and 62 have been replaced by glutamines, were poorer substrates than hirudin. The mutant in which all four glutamates were substituted, [Gln57,58,61,62]hirudin, was virtually not phosphorylated by either TPK-IIB and TPK-III. Substitution of Glu57 and Glu58 was less deleterious than substitution of the two glutamic acids adjacent to Tyr63: [Gln61,62]hirudin exhibited a 20-fold lower phosphorylation efficiency with TPK-IIB. With TPK-III, however, the Km value of [Gln61,62]hirudin was slightly lower, while the Vmax decreased sixfold. The substitution of Glu62 alone was also more detrimental with TPK-IIB than with TPK-III. The behaviour of a third spleen TPK, named lyn TPK-I and belonging to the src family, was markedly different in that it did not phosphorylate hirudin but exhibited significant activity towards [Gln57,58,61,62]hirudin. Taken together, these data confirm and extend with a protein substrate the results obtained with short model peptides which indicated the stringent substrate requirements of TPK-IIB (and of TPK-III to a lesser extent) for N-terminal acidic residues. In contrast, such residues are deleterious with lyn TPK-I. These observations also support the concept that tyrosine protein kinases recognize specificity determinants situated in the vicinity of the target residue rather than requiring higher-order structural features.

Amino Acid Sequence↗
PubMed1991

Peptides reproducing the phosphoacceptor sites of pp60c-src as substrates for TPK-IIB, a splenic tyrosine kinase devoid of autophosphorylation activity.

TPK-IIB, a spleen tyrosine protein kinase devoid of autophosphorylation activity (Brunati, A. M., and Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457), has been purified to near homogeneity and assayed for its ability to phosphorylate the synthetic peptides EDNEYTA and EPQYQPA reproducing the two conserved phosphoacceptor sites of pp60c-src (Tyr-416 and Tyr-527). While EPQYQPA was phosphorylated with low efficiency (Km = 16.7 mM, Kcat = 14.4), EDNEYTA is an excellent substrate displaying a Km value of 58 microM and a Kcat value of 31.2. The single substitution, in the latter peptide, of the glutamic acid adjacent to the tyrosine by alanine to give EDNAYTA caused a 6-fold increase in the Km. The positive influence on the phosphorylation of the acidic residues at -3 and -4 relative to the tyrosine is indicated by comparison of the kinetic constants for peptides EDAAYAA (Kcat = 4.6, Km 0.325 mM) and QNAAYAA (Kcat 2.4, Km 1.7 mM). Furthermore, when residues in the peptide NEYTA were replaced by alanine, the phosphorylation of the peptides NAYTA and AAYAA, was almost negligible (in terms of Kcat/Km ratio). However, AEYTA, NEYAA and AEYAA were still phosphorylated, albeit less efficiently than NEYTA. The probability that these peptides will adopt a beta-turn is EDNAYTA = EDNEYTA, NAYTA greater than NEYTA, and no predicted beta-turn for AEYTA, NEYAA, and AEYAA. Therefore these results support the concept that an amino-terminal acidic residue(s) is strictly required by TPK-IIB, irrespective of peptide conformation, although a beta-turn may enhance the phosphorylation of those peptides that satisfy this requirement. Two other spleen tyrosine kinases, TPK-I/lyn and TPK-III, both related to the src family, also have a far greater preference for the peptide EDNEYTA over EPQYQPA. However, they can be distinguished from TPK-IIB by their lower affinity for the peptides EDNEYTA and NEYTA and by their different specificity towards the substituted derivatives of NEYTA. TPK-I/lyn, accepts most of the substitutions that are detrimental to TPK-IIB, the triply substituted peptide AAYAA being actually preferred over the parent peptide NEYTA. The substitution of glutamic acid by alanine is also tolerated by TPK-III, although, in contrast to TPK-IIB, the phosphorylation efficiency is drastically decreased by the substitution of the asparagine at position -2.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

The use of phosphopeptides to distinguish between protein phosphatase and acid/alkaline phosphatase activities: opposite specificity toward phosphoseryl/phosphothreonyl substrates.

The four main classes of protein phosphatases (PP-1, 2A, 2B and 2C), although differing in their ability to dephosphorylate phosphopeptide substrates, invariably display a marked preference toward phosphothreonyl peptides over their phosphoseryl counterparts. Conversely, all the acidic and alkaline phosphatases tested so far dephosphorylate phosphoseryl derivatives far more readily than phosphothreonyl ones. This opposite behaviour provides a criterion for discriminating between protein dephosphorylating activity due to authentic protein phosphatases as compared to nonspecific acid and/or alkaline phosphatases. In particular the phosphothreonyl peptides RRATPVA and RRREEETPEEEAA appear to be especially suited for detecting the activity of PP-2C and PP-2A, since they are hardly dephosphorylated by acid and alkaline phosphatases. Conversely, the phosphoseryl peptides SPEEEEE and RRASPVA can provide a sensitive evaluation of the majority of acid and alkaline phosphatases, while being refractory to protein phosphatases.

Acid Phosphatase↗
PubMed1991

A synthetic beta-casein phosphopeptide and analogues as model substrates for casein kinase-1, a ubiquitous, phosphate directed protein kinase.

The phosphopeptide Ser (P)-Ser(P)-Ser-(P)-Glu-Glu-Ser22-Ile-Thr, reproducing the 17-24 segment of beta-casein A2 including the seryl residue (Ser-22) which is targeted by casein kinase-1 was synthesized and used as model substrate for this enzyme. Its phosphorylation efficiency is actually higher than that of intact beta-casein (similar Vmax and 14 microM Km). Conversely the fully dephosphorylated peptide SSSEESIT is not affected by CK-1 to any detectable extent and its glutamyl derivative EEEEESIT displays a more than 50-fold higher Km and a 5-fold lower Vmax as compared to the parent phosphopeptide. The relevance of the individual phosphoseryl residues has been assessed by comparing the phosphorylation efficiencies of the phosphopeptides EESpEESIT, ESpEEESIT and SpEEEESIT: while the first is a substrate almost as good as the tris Ser (P)-peptide (Km = 62 microM), and the third one is almost as poor as EEEEESIT (Km = 1.55 mM), ESpEEESIT displays a intermediate efficiency (Km = 277 microM). These data in conjunction with the finding that the phosphopentapeptide Ser(P)-Ser(P)-Ser(P)-Ser-Ser(P), but neither Ser(P)-Ser(P)-Ser-Ser(P) nor Ser-Ser(P)-Ser(P)-Glu-Glu and Ser-Ala-Ala-Ser(P)-Ser(P), is readily phosphorylated by CK-1, support the concept that CK-1 is a phosphate directed protein kinase recognizing the Ser(P)-X-X-Ser-X and, less efficiently, the Ser(P)-X-X-X-Ser-X motifs.

Adenosine Triphosphate↗
PubMed1991

Phosphotyrosine as a specificity determinant for casein kinase-2, a growth related Ser/Thr-specific protein kinase.

The motif Ser-Ser-Ser-Glu-Glu is readily phosphorylated by casein kinase-2 (CK-2), a growth-related protein kinase whose consensus sequence is Ser(Thr)-Xaa-Xaa-Glu(Asp) [(1990) Biochim. Biophys. Acta 1054, 267-283]. Here we show that phosphotyrosine can replace carboxylic acids as specificity determinant for CK-2 phosphorylation, the phosphotyrosyl peptide Ser-Ser-Ser-TyrP-TyrP actually being a substrate more efficient than Ser-Ser-Ser-Glu-Glu itself both in terms of Km (0.69 vs 2.43 mM) and Vmax. Prior dephosphorylation of phosphotyrosine entirely prevents the subsequent phosphorylation of serine by CK-2. While Ser-Ser-Ser-TyrP-TyrP is a better substrate than Ser-Ser-Ser-SerP-SerP, which in turn is better than Ser-Ser-Ser-Glu-Glu, Ser-Ser-Ser-ThrP-ThrP is a less efficient substrate than Ser-Ser-Ser-Glu-Glu. Thus the order of efficiency of phosphoamino acids as specificity determinants for CK-2 appears to be TyrP greater than SerP much greater than ThrP.

Amino Acid Sequence↗
PubMed1991

Further definition of the substrate specificity of the alpha-herpesvirus protein kinase and comparison with protein kinases A and C.

The pseudorabies virus protein kinase prefers model substrates containing arginyl residues on the amino-terminal side of a target seryl or threonyl residue. We have defined this substrate specificity more precisely in experiments using a new series of synthetic model peptides. When the number of arginyl residues was varied from two to four in substrates of the type RnASVA it was found that peptides with four arginyl residues constituted the best substrates, although the most marked decrease in Km was seen on increasing the number of arginyl residues from two to three. The effect of varying the number of 'spacer' alanyl residues from zero to three was investigated in peptides of the type R4AmSVA, and the peptide with one alanyl residue was found to be the best substrate, making R4X the optimal amino-terminal environment for this enzyme. A similar substrate specificity was observed with the herpes simplex type 1 protein kinase. Protein kinase C was found to have a quite similar substrate preference to the viral enzyme as far as the number and position of the amino-terminal basic residues was concerned; but, unlike the viral protein kinase, it also requires carboxy-terminal basic residues in optimal peptide substrates, and can tolerate the substitution of lysyl for arginyl residues. The cyclic AMP-dependent protein kinase, like the viral enzyme, had favourable kinetic constants for this series of peptides, but differed from the latter in being able to catalyze the phosphorylation of the peptides with two to four arginyl residues with similar efficiency. Studies with the protein, clupeine Y1, as substrate indicated that the pseudorabies virus protein kinase can tolerate arginyl residues on the carboxyl-terminal side of its target residue when there are suitable amino-terminal arginyl determinants. In this respect the virus protein kinase resembled protein kinase C but differed from the cyclic AMP-dependent protein kinase which cannot tolerate such carboxyl-terminal basic residues. The relationship of substrate specificity with model peptides to the ability of the pseudorabies virus protein kinase to phosphorylate proteins in vitro and in vivo is discussed.

Amino Acid Sequence↗
PubMed1991

The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2.

The alpha subunit of eukaryotic protein synthesis initiation factor (eIF-2 alpha) is phosphorylated at a single serine residue (Ser51) by two distinct and well-characterized protein kinase, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51-Arg-Arg-Arg-Ile-Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two-dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP-dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both 'sites' in clupeine Y1 and salmine A1, whereas HCR acted only on the N-terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C-terminal clusters of arginines on the 'target' serine residue, provided that they were present at positions +3 and/or +4 relative to Ser51. However, peptides containing only N-terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF-2 alpha and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.

Amino Acid Sequence↗
PubMed1991

Stimulation by NaCl, polylysine and heparin of two forms of spleen tyrosine protein kinase immunologically related with the protein expressed by lyn oncogene.

The previously isolated spleen tyrosine protein kinase, conventionally termed TPK-IIA, displaying activation by either positively or negatively charged polyelectrolytes has been further characterized. TPK-IIA is immunologically related with the tyrosine protein kinase encoded by the lyn gene, a member of src subfamily and is dramatically activated by very high NaCl concentration. The stimulatory effects of NaCl and polylysine, which are not additive, are accounted for by increased Vmax values, the Km being virtually unchanged, suggesting that both effectors probably interact with the same site(s). Stimulation of TPK-IIA by heparin appears to be partially additive to that promoted by NaCl and possibly occurring through a different mechanism. The NaCl activatory effect correlates with the electrolytic nature of synthetic peptides used as substrates, being much more consistent with neutral peptides as compared with acidic ones. Of the other three spleen tyrosine protein kinases, TPK-I shows similar biochemical and immunological features, suggestive of close relatedness with TPK-IIA, while TPK-IIB and TPK-III are neither related with the lyn protein nor with the products of three other oncogenes of the src subfamily, namely lck, hck and fyn.

Animals↗
PubMed1990

Different specificities of spleen tyrosine protein kinases for synthetic peptide substrates.

20 synthetic peptides, each of which includes a tyrosyl residue flanked by either neutral or acidic amino acids in different proportions and at variable positions, have been employed as model substrates for investigation of the site specificity of three tyrosine protein kinases previously isolated from spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and conventionally termed TPK-I, TPK-IIB and TPK-III. Comparison of the phosphorylation efficiencies shows that each tyrosine protein kinase is considerably different from the others in both the stringency and the nature of its specificity determinants. By considering, in particular, the kinetic constants obtained with the pentapeptides AAYAA, EEYAA, AEYAA, EAYAA, with the tetrapeptides AYAA and EYAA and with the tripeptides AYA and EYA, it turns out that N-terminal acidic residue(s) are only essential with TPK-IIB for efficient phosphorylation with multiple residues displaying a synergistic effect. The very similar Km (130 microM) but 14-fold-different Vmax values with YEEEEE vs. EEEEEY indicate that an N-terminal rather than C-terminal location of acidic residues is required for a high phosphorylation rate with, though not for binding to TPK-IIB. Acidic residues decrease the phosphorylation rate with TPK-I, a kinase related to the src family which is immunologically indistinguishable from the lyn TPK; they are nearly ineffective, however, with TPK-III, the least specific of the tyrosine protein kinases, which exhibits appreciable activity toward tripeptides and dipeptides like GAY and AY which are not significantly affected by TPK-I and TPK-IIB. While the peptide substrate specificity of TPK-I is similar to that of TPK-IIA, a spleen tyrosine protein kinase previously considered [Brunati, A. M., Marchiori, F., Ruzza, P., Calderan, A., Borin, G. & Pinna, L. A. (1989) FEBS Lett. 254, 145-149], the remarkable requirement of TPK-IIB alone for acidic peptides may suggest the involvement of this enzyme, which is also unique in its failure to autophosphorylate, in the phosphorylation of the highly conserved and quite acidic phosphoacceptor sites of the src family protein kinases.

Animals↗
PubMed1990

Phosphorylation of src-phosphopeptides by casein kinases-1 and -2: favourable effect of phosphotyrosine.

The synthetic phosphotyrosyl tridecapeptide H-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-Gln-Gly-OH, reproducing a major phosphoacceptor site of protein tyrosine kinases of the src-family, can be phosphorylated at Thr-9 by both casein kinases -1 and -2. Its shorter derivative H-Asn-Glu-Tyr(P)-Thr-Ala-OH is not affected by casein kinase-1 while representing a substrate as good as the tridecapeptide for casein kinase-2. The unphosphorylated analogue H-Asn-Glu-Tyr-Thr-Ala-OH, however, is a much poorer substrate, and no significant phosphorylation could be observed of its O-methyl ether derivative H-Asn-Glu-Tyr(Me)-Thr-Ala-OMe. These data on one side corroborate the concept that casein kinase-1 recognizes residues located on the C-terminal edge of acidic stretches, providing, on the other, the evidence that phosphotyrosyl side chains can act as specificity determinants for casein kinase-2.

Amino Acid Sequence↗
PubMed1990

Phosphorylation of ornithine decarboxylase in intact erythroleukemia cells.

32P-labeled ornithine decarboxylase was isolated by immunoprecipitation from murine erythroleukemia cells incubated in a medium containing [32P]ortophosphoric acid. Analysis of immunoprecipitate by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a radiolabeled band, which corresponded to the position of mouse ornithine decarboxylase, phosphorylated in vitro by casein kinase-2. A preparation of casein kinase-2 purified from nuclei of erythroleukemia cells could also phosphorylate mouse ornithine decarboxylase.

Animals↗
PubMed1990

Synthetic peptides as model substrates for the study of the specificity of the polycation-stimulated protein phosphatases.

The substrate specificity of the different forms of the polycation-stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg-dependent (type 1) phosphatase (AMDC) was investigated, using synthetic peptides phosphorylated by either cyclic-AMP-dependent protein kinase or by casein kinase-2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N-terminal (Arg) basic residues. In contrast with the latter, the AMDC phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N-terminal basic residues also favor the dephosphorylation of otherwise almost inert substrates by the AMDC phosphatase. Hence, while the dephosphorylation of Thr(P) substrates by the PCS phosphatases is highly favored by the nature of the phosphorylated amino acid, phosphatase activity toward Ser(P)-containing peptides may require specific determinants in the primary structure of the phosphorylation site.

Amino Acid Sequence↗
PubMed1990

The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin.

Recombinant hirudin (hirudin), which lacks the sulphate group on Tyr-63, has a tenfold-reduced affinity for alpha-thrombin. Incubation of recombinant hirudin with [gamma-32P]ATP and protein tyrosine kinase III from spleen resulted in incorporation of radioactivity into the protein. Phosphatohirudin was purified to homogeneity (overall yield 5%) and shown to contain 1 mol phosphate/mol protein, as a phosphotyrosyl residue at position 63. The kinetics of the inhibition of human alpha-thrombin by phosphatohirudin were determined. It was found that the introduction of the negatively charged phosphate had fully restored the affinity of recombinant hirudin for alpha-thrombin to the level of the wild-type sulphatohirudin. The inhibition constant of phosphatohirudin was 18 fM compared with 20 fM for that of sulphatohirudin. Moreover, the values for the on- and off-rate constants of both forms of hirudin were indistinguishable.

Adenosine Triphosphate↗