Biomedical subjects

M Prorok

Publications and source records attributed to M Prorok.

At least 37 records · Page 2Linked to original sources

PubMed1997

Serine-578 is a major phosphorylation locus in human plasma plasminogen.

It has been reported that human plasminogen (HPg) exists in plasma in a phosphorylated form. We now document that both major glycoforms of plasma HPg contain a phosphoserine residue in their latent protease chains, as revealed by quantitative protein phosphate determinations and 31P-NMR analysis. The sequence location of the phosphoserine residue was established by time-of-flight matrix-assisted laser desorption ionization with delayed extraction mass spectrometric analysis of peptides resulting from complete tryptic and cyanogen bromide digests of the latent protease chain of HPg. Confirmation of the presence of organic phosphate in the identified peptide was obtained by determination of the resulting mass shift after treatment of the peptide with alkaline phosphatase. The data show that Ser578 is a major phosphorylation site in HPg.

Amino Acid Sequence↗

PubMed1997

High-level secretion in Pichia pastoris and biochemical characterization of the recombinant kringle 2 domain of tissue-type plasminogen activator.

The kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been expressed in Pichia pastoris cell lines GSI 15 and KM71. This construct contained a hexahistidine sequence at the C-terminus of the kringle to aid in purification by immobilized metalion-affinity chromatography. The exact amino acid sequence of the isolated kringle was EAEAYV-[K2tPA]SR(H)6, where [K2tPA] represents amino acid sequence residues C1-C82 of the kringle domain (residues 180-261 of tPA). The clones of the yeast transformants provided large amounts of the recombinant (r)-[K2tPA]-containing polypeptide at levels that allowed ready purification of several hundred mg from shake flasks and near-gram levels from a high-biomass fermenter. Purification of the kringle domain directly from cell-conditioned media was accomplished in a single step by either immobilized Ni(+)-affinity chromatography or lysine-Sepharose affinity chromatography. N-linked glycans were present on approx. 30% of this yeast-expressed material, at N5 of the kringle (corresponds to N11 of the particular construct, N184 of full-length tPA). The expressed recombinant kringle recognized a conformation-specific monoclonal antibody generated against tPA that is directed to the K2 domain of the protein, interacted properly with various omega-amino acid ligands, and showed signature conformational properties when studied by differential scanning calorimetry and high-resolution 1H-NMR. The results demonstrate that the P. pastoris system can be employed to obtain large amounts of secreted and properly folded kringle domains.

Kringles↗

PubMed1996

Calcium binding properties of synthetic gamma-carboxyglutamic acid-containing marine cone snail "sleeper" peptides, conantokin-G and conantokin-T.

Total chemical synthesis of two Conus-derived peptides, conantokin-G (con-G), a 17-residue polypeptide containing five residues of gamma-carboxyglutamic acid (Gla), and conantokin-T (con-T), a 21-residue polypeptide possessing four residues of Gla, was accomplished. Calcium binding isotherms were obtained for each peptide, and these differed considerably from each other. The binding isotherm for con-G was complex and could only be fit to degenerate models involving multiple Ca2+ binding sites. The data for Ca2+ binding to con-T was uniquely fit to a simple one-site model. In the case of con-G, circular dichroism (CD) studies revealed a polypeptide without observable alpha-helicity in the absence of Ca2+ and a dramatic shift to a high degree of alpha-helix at saturating Ca2+ concentrations. In contrast, apo-con-T possessed significant alpha-helical structure, and saturation with Ca2+ produced a less substantial change in its alpha-helical content. Titrations with Ca2+ of the change in alpha-helical content of con-T produced a C50 value for Ca2+ that was essentially the same as its Kd from direct binding studies, demonstrating that occupancy of the single macroscopic binding site resulted in the conformational change. Similar titrations with con-G provided a C50 value in concert with the Kd for binding of Ca2+ to this peptide. Moreover, in agreement with these particular Ca(2+)-induced structural changes, gel filtration analyses demonstrated significantly reduced hydrodynamic volumes of both of these polypeptides after saturation of their apo forms with Ca2+, with con-G showing a more pronounced change than con-T. One-dimensional H-NMR spectra showed both line broadening and changes in chemical shifts of several peptide amide proton resonances after addition of Ca2+ to con-G, again suggestive of a large Ca(2+)-induced conformational change in this polypeptide. A derivative of con-G was synthesized with all amino acids present in the D-configuration (D-con-G). This variant peptide displayed Ca2+ binding isotherms nearly identical to those of con-G and underwent a Ca(2+)-induced conformational change very similar to that of con-G. Intracranial injections of con-G and con-T in young (< 2 weeks) and older (3-4 weeks) mice produced the expected "sleep-like" and hyperactive effects, respectively. The variant, D-con-G, was inactive in these assays. These studies demonstrate that synthetic con-G and con-T possess their expected bioactivities and undergo large and defined conformational alterations in the presence of Ca2+. We propose that binding of Ca2+ to these polypeptides contributes to their ability to adopt a defined conformation, and this divalent cation-dependent conformation is necessary for their neuroactivities.

1-Carboxyglutamic Acid↗

PubMed1996

The entire gamma-carboxyglutamic acid- and helical stack-domains of human coagulation factor IX are required for optimal binding to its endothelial cell receptor.

The minimal region of the gamma-carboxyglutamic acid (Gla) domain of human factor (f) IX that interacted with its putative bovine aortic endothelial cell (BAEC) receptor was examined by chemical synthesis of peptides with sequence counterparts in this region of the protein, and assessment of their relative abilities to compete with fIX for receptor binding. We found that IC50 values (total peptide concentrations needed to achieve 50% inhibition of binding of [125I]-fIX to BAEC) were ca. 18 nM for unlabeled fIX and 23 nM for the peptide consisting of the entire Gla domain/helical stack (HS) region (residues 1-47) of fIX. The peptide containing only the Gla domain of fIX (residues 1-38) displayed an IC50 value of > 500 nM for this same competitive binding, whereas peptides containing sequences present in positions 1-14 and 1-24 of the Gla domain of human fIX did not significantly compete with [125I]-fIX for BAEC binding. We conclude that whereas a specific receptor recognition element is present within residues 1-14 of fIX, as has previously been concluded by others and by us, full expression of this epitope requires its presence within the entire Gla domain and HS for proper folding. All determinants for proper folding of fIX that lead to BAEC receptor binding appear to be present within these two domains.

1-Carboxyglutamic Acid↗

PubMed1995

Hydrophobic amino acid residues of human anticoagulation protein C that contribute to its functional binding to phospholipid Vesicles.

The contributions to functional phospholipid (PL) binding of the cluster of amino acid side chains of human protein C (PC) comprising F4, L5, and L8 have been assessed by construction of mutants of PC and activated protein C (APC) designed wherein a hydrophilic side chain replaced the wild-type hydrophobic groups at these positions. The PL-dependent plasma-based anticoagulant activities of [F4Q]-r-APC and [L8Q]r-APC were severely reduced to 5% and < 2%, respectively, of wild-type r-APC. Activity losses of the mutants toward inactivation of coagulation factor VIII, measured in the complete in vitro tenase system, have also been observed. As evidenced through Ca(2+)-induced intrinsic fluorescence changes, both [F4Q]r-PC and [L8Q]r-PC were able to adopt Ca(2+)-dependent conformations that appeared similar to that of wtr-PC, ruling out shortcomings associated with such Ca(2+)-induced transitions as the basis for their anticoagulant activity losses. However, despite this, [L8Q]r-PC showed greatly defective macroscopic binding properties to PL vesicles, as did to a lesser extent [F4Q]r-PC. These findings were similar to those reported previously for [L5Q]r-PC/APC [Zhang, L., & Castellino, F. J. (1994) J. Biol. Chem. 269, 3590-3595]. We thus propose that the PL-dependent activity losses of these mutants are related to their suboptimal binding to PL or to their misorientation on the PL surface leading to poor alignment of the active sites of the r-APC mutants with the complementary cleavage sites on fVIII/fVIIIa and fV/fVa.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1995

Binding of calcium to individual gamma-carboxyglutamic acid residues of human protein C.

Selectively labeled polypeptides comprising the gamma-carboxyglutamic acid (Gla) domain (GD) and helical stack (HS) regions of human protein C (PC), and consisting of amino acid residues 1-47, have been chemically synthesized and their Ca2+ binding properties assessed by [13C]-NMR methods. A total of nine such polypeptides have been studied, each containing one of the Gla residues fully enriched with [13C] at its two gamma-carboxylate carbon atoms. Additions of Ca2+ resulted in readily measurable [13C] chemical shifts, titrations of which were used to obtain apparent dissociation constants for each Gla residue in the presence of all other such residues. The Ca2+ titration data obtained on each of the nine polypeptides showed that Gla residues 6, 16, 25, and 26 were involved in the higher affinity Ca2+ binding sites, whereas the remaining Gla residues, viz., 7, 14, 19, 20, and 29, coordinated Ca2+ more weakly. The results are consistent with conclusions drawn from functional studies obtained with site-directed mutations of individual Gla residues and with the structural model of the GD/HS of human PC. In these cases, Gla residues 6, 16, and 26 served as coordination loci for internally located Ca2+ ions, and GD-related Ca(2+)- and PL-dependent properties of PC and activated PC were dependent on the integrity of these Gla residues.

1-Carboxyglutamic Acid↗

PubMed1994

Three-dimensional structure of chymotrypsin inactivated with (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane: implications for the mechanism of inactivation of serine proteases by chloroketones.

The reaction of enantiomerically pure (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane with gamma-chymotrypsin was studied as a probe of the mechanism of inactivation of serine proteases by peptidyl chloroalkanes. It was determined crystallographically that the peptidyl chloroethane alkylates His57 with retention of configuration at the chiral center, indicating a double displacement mechanism. We think it likely that a Ser195-epoxy ether adduct is an intermediate on the inactivation pathway, although other possibilities have not been disproven. Kinetic data reported by others [Angliker et al. (1988) Biochem. J. 256, 481-486] indicate that the epoxy ether intermediate is not an irreversibly inactivated form of enzyme [a conclusion confirmed experimentally (Prorok et al. (1994) Biochemistry 33, 9784-9790)] and that both ring closure of the tetrahedral intermediate to form the epoxy ether and ring opening by His57 partially limit the first-order rate constant for inactivation, ki. The peptidyl chloroethyl derivative adopts a very different active site conformation from that assumed by serine proteases inactivated by peptidyl chloromethanes. Positioning the chloroethyl derivative into the conformation adopted by chloromethyl derivatives would cause the extra methyl group to make a bad van der Waals contact with the inactivator P2 carbonyl carbon, thereby preventing the formation of the invariant hydrogen bond between the inactivator P1 amide nitrogen and the carbonyl group of Ser214. We conclude that the unusual conformation displayed by the chloroethyl derivative is caused by steric hindrance between the extra methyl group and the rest of the inactivator chain.

Amino Acid Chloromethyl Ketones↗

PubMed1994

Chloroketone hydrolysis by chymotrypsin and N-methylhistidyl-57-chymotrypsin: implications for the mechanism of chymotrypsin inactivation by chloroketones.

We have examined the reaction of N-(benzyloxycarbonyl)-L-alanyl-L-glycyl-L-phenylalanyl chloromethyl ketone (ZAGFCMK) with chymotrypsin (Cht) and have found that, in addition to irreversible alkylation of the enzyme, some of the corresponding hydroxymethyl ketone is produced. For each molecule of hydroxy ketone formed, 3.6 molecules of chymotrypsin are inactivated. Chloroketone hydrolysis is also observed with chymotrypsin methylated at N-3 of the active site histidine (MeCht). The hydrolysis proceeds slowly (k = 0.14 min-1). Alkylation of the modified enzyme was not observed. An initial burst of free chloride is detected during the MeCht-catalyzed hydrolysis. The magnitude of the chloride burst is proportional to the enzyme concentration in an approximate 1:1 stoichiometry and indicates a relatively rapid chloride-releasing step which gives rise to an intermediate which is more slowly converted to hydroxy ketone. We have also investigated both the solution and MeCht-mediated hydrolysis of the S isomer of N-acetyl-L-alanyl-L-phenylalanyl chloroethyl ketone (S-AcAFCEK). We have concluded that the nonenzymatic hydrolysis proceeds with inversion of configuration at the stereocenter, while the enzymatic process occurs with retention of configuration. The two nucleophilic displacements attending the MeCht-mediated hydrolysis of S-AcAFCEK imply the formation of an intermediate, possibly of an epoxy ether, formed by internal displacement of the chloride by the oxyanion of the initially generated enzyme-chloroketone hemiketal adduct.

Alkylation↗

PubMed1993

ATPase-promoting dead end inhibitors of the cAMP-dependent protein kinase.

The cAMP-dependent protein kinase is a bifunctional enzyme, catalyzing the phosphorylation of the serine and threonine residues in peptides and proteins (kinase activity) as well as the phosphorylation of water (ATPase activity). We have found that several peptides, which serve as inhibitors of the kinase reaction, will either maintain or enhance the ATPase reaction catalyzed by the enzyme. Positively charged dipeptides (e.g. Arg-Arg), as well as small guanidino-containing compounds (e.g. guanethidine) block protein kinase activity yet enhance ATPase activity up to 3.5-fold over that exhibited by the enzyme in the absence of these compounds. In contrast, several nonphosphorylatable peptides, whose primary sequences are based on that of a known substrate (i.e. Leu-Arg-Arg-Ala-Ser-Leu-Gly), such as Leu-Arg-Arg-Ala-Ala-Leu-Gly, Leu-Arg-Arg-Ala-Phe-Leu-Gly, and Leu-Arg-Arg-Ala-Tyr-Leu-Gly, have little or no effect on the rate of the kinase-catalyzed hydrolysis of ATP. An exception to the latter observation is Leu-Arg-Arg-Ala-Cys-Leu-Gly, a cysteine-containing peptide that promotes the protein kinase-catalyzed ATPase reaction by 2.2-fold. We have also found that peptides that possess relatively large amino acid side chain moieties immediately following the arginine dyad (i.e. such as Phe, Tyr, Cys, or Asn at Xaa in Leu-Arg-Arg-Xaa-Ala-Leu-Gly) sharply reduce the rate of enzyme-catalyzed ATP hydrolysis. This suggests that in the presence of peptides containing an -Arg-Arg-Ala- sequence, the enzyme-bound gamma-phosphate of ATP is relatively accessible to water. In contrast, when the latter alanine moiety is replaced by a larger residue, access by water to ATP appears to be hindered. These results indicate that certain structural features associated with the substrate or substrate analog have a profound influence on the manner by which these species interact with the protein kinase. Furthermore, the work described herein demonstrates that it is possible to block the physiologically important kinase reaction and simultaneously promote the energetically wasteful ATPase reaction.

Adenosine Triphosphatases↗

PubMed1990

Noncovalent active site interactions enhance the affinity and control the binding order of reversible inhibitors of the cAMP-dependent protein kinase.

The peptides, Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH2, Leu-Arg-Arg-Gln-Ala-Leu-Gly-NH2, and Leu-Arg-Arg-Asn-Ala-Leu-Gly-NH2, serve as active site-directed inhibitors of the cAMP-dependent protein kinase from bovine cardiac muscle. The Asn-containing peptide is a 10-fold more potent inhibitor than its Ala- and Gln-containing counterparts. All three peptides are linear competitive inhibitors versus a peptide-based substrate and uncompetitive inhibitors versus MgATP. The enhanced inhibitory potency of the Asn-peptide, in conjunction with the observed loss of ATP-ase activity of the enzyme in the presence of the inhibitor, suggests that asparagine may serve as a through-space isostere of serine. The uncompetitive inhibition pattern displayed by amide-capped peptides versus MgATP indicates that these species bind in an ordered fashion to the cAMP-dependent protein kinase, with MgATP binding first.

Adenosine Triphosphatases↗

PubMed1989

Multiple arginine residues contribute to the increased efficacy of peptide substrates for the cAMP-dependent protein kinase.

Efficient cAMP-dependent protein kinase substrates typically contain an arginine dyad one amino acid removed from the residue which undergoes phosphorylation (ie. Arg-Arg-X-Ser). However, several naturally occurring protein kinase inhibitors and substrates possess additional basic residues that are proximal to the arginine dyad, implying the presence of either an extended or an additional acidic subsite on the enzyme. In this study, we investigated the substrate efficacy of several multiple arginine-bearing peptides. The most efficient substrate studied, Arg-Arg-Leu-Arg-Arg-Ala-Ser-Leu-Gly, exhibits a nearly eleven-fold increase in kcat/Km relative to Leu-Arg-Arg-Ala-Ser-Leu-Gly. The enhanced kcat/Km is primarily a consequence of a reduced Km. These results suggest that a double arginine dyad, separated by a single amino acid, represents the optimal sequence for basic residues on cAMP-dependent protein kinase substrates.

Amino Acid Sequence↗

PubMed1989

The cyclic AMP-dependent protein kinase from bovine cardiac muscle is a homoserine kinase.

The catalytic subunit of the cAMP-dependent protein kinase from bovine cardiac muscle phosphorylates homoserine in the synthetic peptide Leu-Arg-Arg-Ala-Hse-Leu-Gly. Phosphorylation of the primary alcohol of the homoserine residue was established via NMR spectroscopy. Two-dimensional correlated and nuclear Overhauser effect spectroscopies provided the sequence-specific chemical shift assignments of the substrate peptide and its phosphorylated counterpart. Coupled and decoupled 31P NMR experiments established the presence of phosphate on the homoserine residue. The maximal velocity (6.4 mumol/min.mg) obtained for homoserine-peptide phosphorylation at 12.5 mM Mg2+ compares favorably to the velocities observed for the corresponding serine- (21 mumol/min.mg), threonine- (3.2 mumol/min.mg), and hydroxyproline-peptides (1 mumol/min.mg). However, the Km for homoserine kinase activity is modest (1.3 mM) relative to the Km associated with the phosphorylation of the serine-containing substrate (22 microM). The effect of Mg2+ concentration on the kinetic parameters kcat, Km, and kcat/Km was investigated for both serine- and homoserine-peptides. Both substrates display similar kcat/Km versus [Mg2+] profiles, with the most notable difference that the optimal Mg2+ concentration is higher for the homoserine-containing peptide. In addition, the Km for the serine-peptide was found to be independent of [Mg2+], whereas the Km for the homoserine-peptide was observed to be dependent upon [Mg2+]. These results suggest that the long homoserine side chain may induce an unusually large off rate for the peptide and/or may misalign the hydroxyl moiety in the active site.

Amino Acid Sequence↗

PubMed1989

Intrasubstrate steric interactions in the active site control the specificity of the cAMP-dependent protein kinase.

The cAMP-dependent protein kinase catalytic subunit phosphorylates serine residues more efficiently than threonine residues in synthetic peptides. In marked contrast, both amino acids are phosphorylated at similar rates when contained within the appropriate intact protein substrate. The structural basis for the discriminatory behavior observed in small peptides has been investigated and found to be a result of intrapeptide steric interactions in the vicinity of the threonine alcohol moiety. Leu-Arg-Arg-Gly-Thr-Leu-Gly, which is nearly free of these interactions, is phosphorylated at a rate that is almost comparable to its serine-containing counterpart.

Amino Acid Sequence↗

PubMed1989

Cryopreservation of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase from bovine cardiac muscle.

A method for the cryogenic storage of the cAMP-dependent protein kinase from bovine cardiac muscle is described. The catalytic parameters, kcat, KM, and kcat/KM are used to assess the activity of the enzyme both prior and subsequent to the freeze-thaw cycle. The enzyme is stored in cryogenic vials at -196 degrees C in liquid nitrogen. Complete retention of catalytic activity is dependent upon a rapid and efficient freeze-thaw cycle and the use of morpholinepropanesulfonic acid as the buffer. In addition, this buffer appears to eliminate the KCl- or NaCl-induced damage typically observed for enzymes stored at low temperature in phosphate buffer. As a result, morpholinepropanesulfonic acid may prove to be a more appropriate cryopreservation buffer than phosphate when the presence of salt is required for enzyme solubility or stability.

Animals↗

PubMed1978