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D A Walsh

Publications and source records attributed to D A Walsh.

At least 145 records · Page 8Linked to original sources
PubMed1986

The inhibitor protein of the cAMP-dependent protein kinase-catalytic subunit interaction. Parameters of complex formation.

The formation of a complex between the catalytic subunit of the cAMP-dependent protein kinase and the Inhibitor Protein of this enzyme has been examined by means of nondenaturing gel electrophoresis. Two forms of complex were identified, both containing a 1:1 molar ratio of the component proteins. The formation of the major of the two forms is markedly enhanced by the presence of nucleotide triphosphate and divalent cation. Either Mg2+ or Mn2+ serves to promote complex formation. With Mg2+, only ATP is effective for enhancing complex formation, whereas with Mn2+ complex formation occurs to an equal extent with ATP, GTP, ITP, and adenyl-5'-yl imidodiphosphate. The formation of the two complexes is only minimally dependent upon nucleotide triphosphate. It is suggested that the two types of complex are a result of different species of catalytic subunit. Two principal forms of the complex have been detected occurring maximally in approximately a 2.5:1 ratio. In the accompanying paper (Fletcher, W.H., Van Patten, S.M., Cheng, H-C., and Walsh, D.A. (1986) J. Biol. Chem. 261, 5504-5513), we have described the use of a fluoresceinated derivative of catalytic subunit as a cytochemical probe to localize the Inhibitor Protein and the regulatory subunit of the protein kinase. The integrity of this fluorophore has been further characterized using the method of examining catalytic subunit-Inhibitor Protein interaction delineated here.

Adenosine Triphosphate↗
PubMed1986

A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase.

As an important new reagent for studying the cAMP-dependent protein kinase, a 20-residue peptide has been synthesized that corresponds to the active site of the skeletal muscle inhibitor protein. This synthetic peptide inhibits the protein kinase competitively with a Ki = 2.3 nM; its sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr- Gly-Arg-Arg-Asn-Ala-Ile-His-Asp, is that of a peptide previously reported by us which was derived from the native inhibitor protein by V8 protease digestion (Cheng, H. C., Van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661). Studies with analogues of this peptide show that its high affinity binding to the protein kinase (as also of the inhibitor protein) appears to be due to it mimicking the protein substrate by binding to the catalytic site via the arginine-cluster basic subsite (Formula: see text), and also to a critical contribution from one or more of the 6 N-terminal residues (Formula: see text). The availability of this high affinity synthetic peptide should open up a variety of avenues to probe the cellular actions of cAMP.

Amino Acid Sequence↗
PubMed1986

Influence of HbS levels upon the hematological and clinical characteristics of sickle cell trait.

The variable concentration of HbS in individuals with sickle cell trait led us to study the relationship between HbS level and selected vascular events in 355 hospitalized black men with sickle cell trait. There were significant differences in hemoglobin concentration and mean corpuscular volume found in four groups divided by their HbS level, the lowest proportion of HbS (less than 30%) being associated with the lowest hemoglobin concentration (12.6 g/dl) and MCV (77 fl). The percent HbS did not influence the incidence of pulmonary embolism, thrombophlebitis, myocardial infarction, cerebrovascular accident, or idiopathic hematuria. Our results suggest that HbS level does not influence vascular disease, and while certain hematological alterations occur, they are very unlikely to have any clinical significance. Regardless of the proportion of HbS, sickle cell trait in black men is benign.

Anemia, Sickle Cell↗
PubMed1986

McArdle's disease heterozygotes. Metabolic adaptation assessed using 31P-nuclear magnetic resonance.

Two daughters of a propositus with documented McArdle's disease were shown by enzyme assay, gel electrophoresis, and immunoblotting to be partially deficient in skeletal muscle phosphorylase and, presumably, heterozygous for the trait. Both exhibited only the adult form of the skeletal muscle isozyme. By 31P-nuclear magnetic resonance, both heterozygotes showed a greater production of acid during fully aerobic exercise than when blood flow was occluded in ischemic exercise. This pattern is in contrast to that of control subjects, where there is significantly greater acid production in ischemic versus aerobic exercise, and distinct from that of phosphorylase-negative patients in which no acid is produced in either circumstance. We suggest that these heterozygotes may have adapted to their diminished phosphorylase by enhancing utilization of plasma glucose. If so, this mechanism could account for the observation that most of the symptoms of McArdle's disease are often manifest only in adulthood. These studies also show that although there are very high concentrations of phosphorylase in skeletal muscle (approximately 2% of the soluble protein), such a high level is essential for normal muscle glycogenolysis.

Adult↗
PubMed1985

An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase.

Digestion with Staphylococcus aureus V8 proteinase of the inhibitor protein of the cyclic AMP-dependent protein kinase results in the sequential formation of three active inhibitory peptides. The smallest active peptide has the sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp . This 20-amino-acid-residue peptide has 20-40% of the activity of the native molecule and a Ki of 0.2 nM. Inhibition, as a minimum, appears to be based upon the inhibitor protein containing the recognition sequences that dictate protein-substrate-specificity. This inhibitory peptide also has sequence homology with the phosphorylation site for a protein kinase other than the cyclic AMP-dependent enzyme.

Amino Acid Sequence↗
PubMed1985

Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase.

A limiting requirement for substrate specificity of the cAMP-dependent protein kinase is the presence of one or two basic residues located to the N-terminal side of the target substrate serine. Furthermore, circular dichroic (CD) studies have shown that binding of protein substrate involves a series of at least two independent conformational changes in the enzyme, each of which is initiated by a recognition signal on the substrate protein. The present study attempts to elucidate further the complete sequence of enzyme/ligand interactions by using the synthetic substrate peptide Kemptide and analogues differing from it at crucial points in the sequence: the Ala-peptide, where alanine is substituted for the target serine, and D-Ser-Kemptide, where the target serine is in the D rather than the L configuration. Examination of the effects of binding of these substrates on the intrinsic UV CD of the enzyme and the induced CD in the presence of Blue Dextran has revealed a third step in the substrate/enzyme binding interaction. Although sections of the conformational change at the active site are dependent on the basic subsite and the serine hydroxyl group on the peptide, respectively, the complete conformational change requires that the substrate be bound in random coil conformation. Where this does not occur, the kinetics show that the peptide will not act either as substrate or as inhibitor of the enzyme. Further, the interaction between the serine hydroxyl group and an enzyme tyrosine residue, previously observed, appears to be dependent on the correct orientation as well as the mere presence of the target -OH group.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1985

Subunit phosphorylation and activation of skeletal muscle phosphorylase kinase by the cAMP-dependent protein kinase. Divalent metal ion, ATP, and protein concentration dependence.

This report provides a characterization of the effects of varying the concentrations of Mg2+, ATP, phosphorylase kinase, and the cAMP-dependent protein kinase on the activation and phosphorylation of phosphorylase kinase. The results show the following. (a) The Km for MgATP2- for the cAMP-dependent protein kinase-catalyzed phosphorylation is decreased by increasing Mg2+, probably as a consequence of decreasing the free ATP:MgATP2- ratio and increasing free Mg2+. (b) Whereas beta subunit phosphorylation of phosphorylase kinase plays a prominent role in determining its activity, alpha subunit phosphorylation can also modulate activity. (c) The phosphorylation of the alpha subunit, which occurs following the initial cAMP-dependent phosphorylation of the beta subunit, is catalyzed by the cAMP-dependent protein kinase and is not a consequence of EGTA-insensitive (or EGTA-sensitive) autophosphorylation occurring as a result of the enhanced phosphorylase kinase activity. (d) The relationship between subunit phosphorylation and phosphorylase kinase activation is complex and particularly dependent upon concentrations of cAMP-dependent protein kinase and phosphorylase kinase in the activation reaction. The data suggest the possibilities that the pathway of phospho-intermediates involved in the activation process probably varies with the activation conditions, that the efficacy of a specific site to be covalently modified is dependent upon the phosphorylation status of other sites, and that the effect of phosphorylation in regulating activity may also be dependent on the phosphorylation status of other sites. It is clear from the data that the activation process for phosphorylase kinase can be very complex, and it is possible that this complexity might have significant physiological ramifications.

Adenosine Triphosphate↗
PubMed1985

A fast-twitch oxidative-glycolytic muscle with a robust inward calcium current.

This report describes the histochemical and physiological properties of a rat skeletal muscle with a robust activity-dependent slow inward Ca2+ current. The muscle, the flexor digitorum brevis (FDB), is a small plantar flexor from the hindfoot. It is a homogeneous muscle consisting of approximately 90% fast-twitch oxidative-glycolytic (type IIA) fibers. Stimulation of the FDB with repetitive stimulus trains (30 or 50 Hz for 330 ms, 1 train/s for 2-5 min) produced a slow increase in the base-line or resting tension of the muscle between trains. This progressive increase in resting tension appears to be due to the activation of voltage-dependent slow Ca2+ channels, since it could be eliminated (i) by stimulating the muscle in a medium containing 2 mM EGTA and without Ca2+, and (ii) by the addition of either Co2+ or verapamil. The presence of a slow current may be associated with an increase in K+ efflux as stimulation continues, and with a prolongation of relaxation time. We also propose that the slow Ca2+ current may contribute to the allosteric activation of phosphorylase kinase during muscle activity. The FDB provides an excellent preparation to investigate the regulation of muscle metabolism by intra- and extra-cellular Ca2+ during exercise.

Action Potentials↗
PubMed1984

Colon carcinoma and diabetes mellitus.

A significantly increased occurrence of overt diabetes mellitus was found among colon or rectal carcinoma patients by comparison with that found among lung carcinoma or hip fracture populations in two hospitals.

Adolescent↗
PubMed1984

The control of phosphorylase kinase phosphatase activity by polycations and the deinhibitor protein.

The dephosphorylation of phosphorylase beta kinase by the activated ATP, Mg-dependent protein phosphatase, which is highly specific for the beta-subunit, is stimulated by the deinhibitor protein which neutralizes the effect of inhibitor-1 and the modulator protein on the phosphatase. The specific dephosphorylation of the alpha-subunit of phosphorylase beta kinase by a "latent" protein phosphatase isolated from vascular smooth muscle is stimulated by histone H1 but not affected by the deinhibitor protein. These observations show that there is no strict correlation between the insensitivity of a protein phosphatase to inhibitor-1 or modulator protein and the dephosphorylation of the alpha-subunit of phosphorylase beta kinase.

Adenosine Triphosphate↗
PubMed1984

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase.

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1984

Intrapartum fetal heart rate monitoring as a predictor of fetal distress and immediate neonatal condition in low-birth weight (less than or equal to 1,800 grams) infants.

Among 1,318 live born infants delivered in our institution during a 120-day period, 1,025 (77.8%) were monitored electronically. Of the 1,025 monitored infants, 89 were of low birth weight (less than or equal to 1,800 gm) and were admitted to the neonatal intensive care unit. Twenty-seven (30%) of these had abnormal fetal heart rate tracings. The remaining 62 (70%) had normal fetal heart rate tracings. Of the 27 low-birth weight neonates with an abnormal fetal heart rate tracing, 24 (89%) were asphyxiated, whereas of those 62 low-birth weight infants with a normal fetal heart rate tracing, only nine (14%) had asphyxia (p less than 0.001). Of the 27 low-birth weight neonates with abnormal fetal heart rate tracings, 20 (74%) developed hyaline membrane disease, whereas of the 62 low-birth weight neonates with normal fetal heart rate tracings, 10 (16%) developed hyaline membrane disease (p less than 0.001). The results of this study suggest that electronic fetal monitoring provides a specific and sensitive method for identifying those low-birth weight infants who are at high risk for asphyxia and hyaline membrane disease.

Asphyxia Neonatorum↗
PubMed1984

The effects of phenylalanine on cultured rat embryos.

The offspring of mothers with untreated classic phenylketonuria (PKU) have shown a high frequency of microcephaly, mental retardation, pre- and postnatal growth retardation, and birth defects. The aim of this study was to determine whether phenylalanine (phe) is the teratogenic agent in maternal PKU. To observe the direct effects of phe on organogenesis, embryos of 9.5-day pregnant rats were cultured for 48 h in the presence of phe at concentrations of 0.1 to 6.0 mM. Within this range no morphological abnormalities occurred in exposed embryos, when compared to control embryos. However there was a reduction (P less than or equal to 0.05) in embryonic protein content and somite number at the highest concentration of phe (6.0 mM). This does not preclude the longer-term effects of phe at later stages of gestation. To examine phe transport into the embryo in response to elevated serum phe levels, 3H-phe uptake studies were undertaken. These showed that 3H-phe from the culture serum is incorporated rapidly into the free amino acid pools and embryonic protein. At serum concentrations of 1.4 mM or higher, phe saturation occurs in the cellular pools of the embryo. Amino acid analysis of the exocoelomic fluid showed that when embryos were cultured for 48 h in serum containing 3.45 mM phe, the total amino acid concentration was maintained near the control levels (16 mM). Of this, 27% (4.26 mM) was contributed by phe, and all other amino acids, except methionine, were decreased with respect to control levels.

Animals↗
PubMed1984

Characteristics of the adenohypophyseal Ca2+-phospholipid-dependent protein kinase.

The Ca2+-phospholipid-dependent protein kinase, initially described by Takai et al. (J. Biol. Chem. 254, 3692-3695, 1979), has been identified in the anterior pituitary gland of the rat and sheep. The enzyme is essentially undetectable in initial cell extracts but marked activity is manifest following DEAE chromatography, suggesting the potential presence of an endogenous inhibitor of this enzyme. Two forms of this protein kinase exist in both sheep and rat anterior pituitary gland, both of which are similarly dependent upon Ca2+, phosphatidyl serine and diacylglycerol. Several endogenous substrates for this protein kinase have been observed in both the pars distalis and pars tuberalis of the sheep adenohypophysis.

Animals↗
PubMed1984

Antiinflammatory activity of N-(2-benzoylphenyl)alanine derivatives.

A series of N-(2-benzoylphenyl)alanine derivatives were synthesized and tested for antiinflammatory activity in the Evans blue-carrageenan induced pleural effusion assay. The target compounds were envisioned to bind to a receptor site on the cyclooxygenase enzyme by a mechanism first proposed by Appleton and Brown. Of the 21 compounds prepared, two were found to be one-tenth as potent as indomethacin in the pleurisy model and one compound was tested and found to be weakly active in the adjuvant arthritis model.

Animals↗