An inhibitory segment of the catalytic subunit of phosphorylase kinase does not act as a pseudosubstrate.

The C terminus of the catalytic gamma subunit of phosphorylase kinase contains two autoinhibitory calmodulin binding domains designated PhK13 and PhK5. These peptides inhibit truncated gamma(1-300). Previous data show that PhK13 (residues 302-326) is a competitive inhibitor with respect to phosphorylase b, with a K(i) of 1.8 microm. This result suggests that PhK13 may bind to the active site of truncated gamma(1-300). Variants of PhK13 were prepared to localize the determinants for interaction with the catalytic fragment gamma(1-300). PhK13-1, containing residues 302-312, was found to be a competitive inhibitor with respect to phosphorylase b with a K(i) of 6.0 microm. PhK13 has been proposed to function as a pseudosubstrate inhibitor with Cys-308 occupying the site that normally accommodates the phosphorylatable serine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kinetic characterization of this peptide reveals that it does not serve as a substrate but is a competitive inhibitor. Additional variants were designed based on previous knowledge of phosphorylase kinase substrate determinants. Variants were analyzed as substrates and as inhibitors for truncated gamma(1-300). Although PhK13-1 does not appear to function as a pseudosubstrate, several specificity determinants employed in the recognition of phosphorylase b as substrate are utilized in the recognition of PhK13-1 as an inhibitor.

Site-directed mutants of glycogen phosphorylase are altered in their interaction with phosphorylase kinase.

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) produces phosphorylase a, which is active in the absence of AMP. PhK is the only kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this specificity and how these two enzymes recognize each other by studying site-directed mutants of glycogen phosphorylase. All mutants were assayed for changes in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, gamma(1-300). Five mutations (R69K, R69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by gamma(1-300) compared to wild-type phosphorylase b. Five mutations, made at three sites in the amino-terminal tail of phosphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for gamma(1-300), compared to that for phosphorylase b. R16E was the poorest substrate for gamma(1-300), giving a 47-fold decrease in catalytic efficiency. The amino terminus, and especially Arg 16, are very important factors for recognition of phosphorylase by gamma(1-300). A specific interaction between Lys 11 of phosphorylase and Glu 110 of gamma(1-300) was also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.

Arginine to citrulline replacement in substrates of phosphorylase kinase.

Synthetic peptides based on residues 9 to 18 of glycogen phosphorylase were prepared containing citrulline at position 10 or 16, or at both positions 10 and 16. The peptides were compared as substrates for a recombinant, truncated form of the catalytic subunit of phosphorylase kinase (residues 1-300). The peptide having citrulline at position 10 was phosphorylated the same as the parent peptide. Both the peptides with a single citrulline at position 16 and with two citrullines were phosphorylated less effectively than the parent peptide; k(cat)/K(m) values were approximately 20% the value with the parent peptide. Incorporation of the second citrulline had little change in the effectiveness of the peptide as a substrate although the kinetic parameters with the citrulline peptides did show differences. The change in peptide phosphorylation did not seem to result from a change in peptide structure. Two-dimensional nuclear magnetic resonance studies of di-citrulline peptide are reported and showed no change in the solution structure of the peptide compared to the parent peptide. Thus, the change in kinetic parameters with the modified peptides seemed an effect of arginine replacement and was likely a consequence of the loss of charge inasmuch as the size of arginine and citrulline are similar. Arginine-16 was concluded to be more important for phosphorylase kinase recognition than arginine-10. These findings were consistent with the earlier studies using alanine replacement of arginine in synthetic peptides as substrates for the holoenzyme form of phosphorylase kinase.

Substrate and inhibitor recognition of protein kinases: what is known about the catalytic subunit of phosphorylase kinase?

Although much can be learned about the specificity of protein kinases from studies with peptide substrates, the question remains, how do kinases recognize their three-dimensional protein substrates? Information derived from such studies provides further understanding of substrate recognition and can facilitate the design of specific protein kinase inhibitors. Phosphorylase kinase (PhK) catalyzes the phosphorylation of phosphorylase b (phos. b) to form the active phosphorylase a. No other protein kinase can duplicate this reaction. Why? To probe this question and establish what features in the protein are important for substrate binding and product release, mutants of phos. b have been studied. This report shows how mutations change the properties of the protein substrate and the ability of these mutants to be phosphorylated by PhK and other kinases. Action of protein kinases on their substrates is often regulated by autoinhibitory segments. The C-terminus of the catalytic gamma-subunit of PhK contains two inhibitory sites overlapping two calmodulin-binding regions. These two peptide segments resemble sequences in phos. b and may explain why peptides of these regions are potent inhibitors of PhK. We will show results with peptide inhibitors, using various expressed forms of the catalytic subunit, which describe their modes of interaction and mechanisms of inhibition. Metal ions can change molecular interactions. With PhK, Mn2+ facilitates the use of GTP as a phosphoryl group donor and greatly increases phosphorylation of a tyrosine residue in angiotensin II. This implies that the spatial arrangement of specificity determinants can be manipulated so that PhK can utilize other substrates.

A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.

Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience.

OBJECTIVE: To determine the source of and describe a large outbreak of Escherichia coli O157:H7 infections in Washington State. DESIGN: Case-control study; environmental investigation; provider-based surveillance for E coli O157:H7 infections. SETTING: Chain of fast-food restaurants, hospitals, physician offices, local laboratories, and local health departments. PARTICIPANTS: Patients with diarrhea and neighborhood controls. A case was defined as diarrhea with culture-confirmed E coli O157:H7 infection or postdiarrheal hemolytic uremic syndrome (HUS) occurring from December 1, 1992, through February 28, 1993, in a Washington State resident. Controls were age- and neighborhood-matched friends of the first 16 case patients. INTERVENTIONS: Announcement to the public; recall of implicated hamburger lots. MAIN OUTCOME MEASURE: Abatement of outbreak due to E coli O157:H7. RESULTS: Infection was associated with eating at a fast-food chain (chain A) in the 10 days before symptoms began. Twelve (75%) of 16 case patients but no controls had eaten at chain A (matched odds ratio undefined; lower 95% confidence interval, 3.5; P < .001). In total, 501 cases were reported, including 151 hospitalizations (31%), 45 cases of HUS (9%), and three deaths. Forty-eight patients (10%) had secondary infections. Of the remaining 453 patients (90%), 398 (86%) reported eating at a Washington chain A restaurant; 92% of them reported eating a regular hamburger. The pulsed-field gel electrophoresis pattern of the E coli O157:H7 strains isolated from all regular hamburger lots of a single production date shipped to Washington was identical to that of the strains isolated from patients. Ten (63%) of 16 regular hamburgers cooked according to chain A policy had internal temperatures below 60 degrees C. Public health action removed more than 250,000 potentially contaminated hamburgers, preventing an estimated 800 cases. CONCLUSIONS: This E coli O157:H7 outbreak, the largest reported, resulted from errors in meat processing and cooking. Public health surveillance through state-mandated reporting of E coli O157:H7 infection as is carried out in Washington State was critical for prompt outbreak recognition and control. Measures should be developed to reduce meat contamination. Consumers and food service workers should be educated about cooking hamburger meat thoroughly.

Anisotropy in alphanumeric reproduction tolerances.

The relative importance of spatial image characteristics in the reproduction of alphanumeric characters has been examined, and it is concluded that significant anisotropy exists. At low distortion levels, horizontal image degradation is somewhat less objectionable than vertical degradation, but at higher levels vertical distortion is significantly less objectionable. The upper and lower portions of characters are found to be much more important for legibility than are the central portions. Upper portions of characters carry more legibility information than the lower portions. The implications of these findings on image transmission systems are discussed.