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D B Sacks

Publications and source records attributed to D B Sacks.

18 recordsLinked to original sources
PubMed1992

Calmodulin-specific monoclonal antibodies inhibit DNA replication in mammalian cells.

The involvement of calmodulin in the proliferation of Chinese hamster embryo fibroblast cells has been studied with a specific monoclonal antibody to calmodulin. We observed that calmodulin levels increase 2-fold in the late G1 period in these cells, and this coincides with the increase in DNA polymerase alpha activity as the cells progress synchronously from a quiescent state in the G1 to the S phase. However, there is a concurrent 10-fold enhancement of thymidine kinase activity, which is tightly coupled to the entry of cells into the S phase. Incubation of permeabilized S-phase cells with calmodulin-specific murine monoclonal antibody resulted in a dose-dependent inhibition of DNA replication. This inhibitory effect of anti-calmodulin antibodies on DNA replication is completely reversed by the addition of exogenously purified calmodulin. These observations provide evidence for the involvement of calmodulin in DNA replication and, therefore, in cell proliferation during the S phase.

Animals↗
PubMed1992

Casein kinase II-catalysed phosphorylation of calmodulin is altered by amino acid deletions in the central helix of calmodulin.

Calmodulin is phosphorylated by casein kinase II on Thr-79, Ser-81, Ser-101 and Thr-117. To determine the consensus sequences for casein kinase II in intact calmodulin, we examined casein kinase II-mediated phosphorylation of engineered calmodulins with 1-4 deletions in the central helical region (positions 81-84). Total casein kinase II-catalyzed phosphate incorporation into all deleted calmodulins was similar to control calmodulin. Neither CaM delta 84 (Glu-84 deleted) nor CaM delta 81-84 (Ser-81 to Glu-84 deleted) has phosphate incorporated into Thr-79 or Ser-81, but both exhibit increased phosphorylation of residues Ser-101 and Thr-117. These data suggest that phosphoserine in the +2 position may be a specificity determinant for casein kinase II in intact proteins and/or secondary structures are important in substrate recognition by casein kinase II.

Amino Acid Sequence↗
PubMed1992

Insulin-stimulated phosphorylation of calmodulin.

Calmodulin is phosphorylated in vitro by the insulin-receptor tyrosine kinase and a variety of serine/threonine kinases. Here we report that insulin stimulates the phosphorylation of calmodulin on average 3-fold in intact rat hepatocytes. Although calmodulin is constitutively phosphorylated, insulin increases phosphate incorporation into serine, threonine and tyrosine residues. We demonstrate that casein kinase II, an insulin-sensitive kinase, phosphorylates calmodulin in vitro on serine/thyronine residues (Thr-79, Ser-81, Ser-101 and Thr-117). The ability of the insulin receptor to phosphorylate calmodulin that has been pre-phosphorylated by casein kinase II is enhanced up to 35-fold, and the sites of phosphorylation on calmodulin are shifted from tyrosine to threonine and serine. These observations, obtained with a new specific monoclonal antibody to calmodulin, confirm that insulin stimulates calmodulin phosphorylation in intact cells. The observation that calmodulin is phosphorylated in vivo, coupled with the recent demonstration that phosphocalmodulin exhibits altered biological activity, strongly suggests that phosphorylation of calmodulin is a critical component of intracellular signalling.

Amino Acid Sequence↗
PubMed1992

Phosphorylation by casein kinase II alters the biological activity of calmodulin.

Calmodulin is the major intracellular Ca(2+)-binding protein, providing Ca(2+)-dependent regulation of numerous intracellular enzymes. The phosphorylation of calmodulin may provide an additional mechanism for modulating its function as a signal transducer. Phosphocalmodulin has been identified in tissues and cells, and calmodulin is phosphorylated both in vitro and in intact cells by various enzymes. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreases its ability to activate both myosin-light-chain kinase and cyclic nucleotide phosphodiesterase. For myosin-light-chain kinase the primary effect is an inhibition of the Vmax. of the reaction, with no apparent change in the concentration at which half-maximal velocity is attained (K0.5) for either Ca2+ or calmodulin. In contrast, for phosphodiesterase, phosphorylation of calmodulin significantly increases the K0.5 for calmodulin without noticeably altering the Vmax. or the K0.5 for Ca2+. The higher the stoichiometry of phosphorylation of calmodulin, the greater the inhibition of calmodulin-stimulated activity for both enzymes. Therefore the phosphorylation of calmodulin by casein kinase II appears to provide a Ca(2+)-independent mechanism whereby calmodulin regulates at least two important target enzymes, myosin-light-chain kinase and cyclic nucleotide phosphodiesterase.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Effects of cationic polypeptides on the activity, substrate interaction, and autophosphorylation of casein kinase II: a study with calmodulin.

The effects of basic polypeptides on the ability of casein kinase II to phosphorylate an exogenous substrate (calmodulin) are correlated with steady-state autophosphorylation of the alpha- and beta-subunits of casein kinase II. Polylysine and polyarginine increase autophosphorylation of the alpha-subunit with a concomitant decrease in beta-subunit phosphorylation, while enhancing casein kinase II-stimulated phosphorylation of calmodulin over 100-fold. The highly basic carboxyl terminal segment of the endogenous p21c-Ki-ras has similar effects on the phosphorylation of calmodulin and the alpha- and beta-subunits of casein kinase II. Altering the concentration of cationic polypeptides produces a biphasic effect on the phosphorylation of both calmodulin and the alpha-subunit, which correlate positively with each other but do not correlate with beta-subunit phosphorylation. When the KCl concentration is changed, casein kinase II activity correlates positively only with alpha-subunit phosphorylation. In contrast, the biphasic response of calmodulin phosphorylation by casein kinase II at different Ca2+ concentrations correlates positively with both alpha- and beta-subunit phosphorylation. Therefore, in the presence of basic protein activators, the rate of phosphorylation of a substrate, calmodulin, correlates with steady-state phosphorylation of the alpha-subunit, but not with the beta-subunit under all conditions tested. Endogenous cationic factors may modulate the in vivo activity of casein kinase II and alter the interaction of the enzyme with specific intracellular substrates.

Animals↗
PubMed1991

Monoclonal antibody to calmodulin: development, characterization, and comparison with polyclonal anti-calmodulin antibodies.

Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free calmodulin than for Ca(2+)-calmodulin. Other selected members of the calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism.

Animals↗
PubMed1990

Radial partition fluorescent immunoassay of thyrotropin. Analytic evaluation and clinical correlation.

The authors evaluated the analytic and clinical performance of a sensitive radial partition fluorescent enzyme immunoassay for thyrotropin (TSH) performed on Stratus and compared it with a nonsensitive radioimmunoassay (RIA) method. Sensitivity of 0.15 mIU/L was obtained, and precision, specificity, and linearity were acceptable. A good correlation was observed between the two assays in samples from 311 hospitalized patients (r = 0.976). Stratus TSH results were outside the reference range for 20% of clinically euthyroid patients (n = 126), and 2.4% had undetectable levels. The clinically hyperthyroid group (n = 11) with the exception of one patient had TSH values below 0.2 mIU/L. Only 39% of hypothyroid patients on thyroid hormone replacement (n = 74) had TSH values in the reference range, with 38% and 23% exhibiting low and high values, respectively. All untreated primary hypothyroid patients (n = 8) had elevated TSH concentrations. The authors conclude that this sensitive TSH assay is useful for diagnosing hyperthyroidism when there is a clinical suspicion but cannot be recommended for thyroid screening in hospitalized patients.

Adult↗
PubMed1990

Interference in an automated radial partition fluorescent immunoassay of thyrotropin associated with liver-function abnormalities.

In a previous evaluation of a "sensitive" radial partition fluorescent immunoassay on the Stratus system, thyrotropin (TSH) values exhibited a positive bias in icteric samples when compared with results of a nonsensitive radioimmunoassay. In the present study, we evaluated 366 patients samples to assess whether any biochemical markers of liver function could identify samples for which TSH values would be falsely increased. gamma-Glutamyltransferase and total bilirubin concentrations were unrelated to discrepant TSH values. In contrast, alkaline phosphatase (ALP) was significantly positively correlated with differences in Stratus and RIA TSH concentrations (P less than 0.001). However, this correlation explained only 34% of the observed residual variability around the estimated regression line. On average, the higher ALP values were associated with larger discrepancies between Stratus and RIA TSH values, although several samples with increased ALP did not have falsely increased Stratus TSH values. TSH measurements performed with a Stratus should be interpreted with caution in patients with abnormal biochemical markers of liver function.

Autoanalysis↗
PubMed1989

Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta.

It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin receptor in a Ca2+-dependent manner, whereas it binds to polylysine seemingly by electrostatic interactions. These studies identify calmodulin as a substrate for the highly purified insulin receptor tyrosine kinase of human placenta. They also demonstrate that the basic proteins, which are required for insulin to stimulate the phosphorylation of calmodulin, do so by a direct interaction with calmodulin.

Calmodulin↗
PubMed1989

Effect of basic polycations and proteins on purified insulin receptor. Insulin-independent activation of the receptor tyrosine-specific protein kinase by poly(L-lysine).

Since the studies on tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro suggested that protamine and poly(L-lysine) may activate phosphorylation of the receptor beta subunit [Sacks & McDonald (1988) J. Biol. Chem. 263, 2377-2383], we examined the effects of a variety of basic polycations/proteins and polyamines on insulin receptor kinase activity. The insulin receptor purified from human placental membranes was incubated with each basic polycation/protein or polyamine and assayed for tyrosine-specific protein kinase activity by measuring 32P incorporation into the src-related peptide. At a concentration of 1 microM, poly(L-lysine) and poly(L-ornithine) markedly stimulated kinase activity, whereas poly(L-arginine) and histones H1 and H2B inhibited insulin receptor kinase. In contrast, at a concentration of 1 mM, three polyamines (spermine, spermidine and putrescine) did not alter kinase activity. Poly(L-lysine) and poly(L-ornithine) stimulated the insulin receptor kinase by 5-10-fold at concentrations of 0.1-1 microM. Protamine sulphate also showed a significant stimulatory effect at a concentration of 100 microM. Preincubation of the receptor with poly(L-lysine) or poly(L-ornithine) for 20-60 min resulted in maximal kinase activation. Poly(L-lysine), the most effective activator of the receptor kinase, was used to characterize further the mechanisms of the kinase activation. Poly(L-lysine) activates the insulin receptor kinase by increasing the Vmax. without changing the Km. Poly(L-lysine) markedly stimulates the kinase activity of insulin receptor preparations that have lost both basal kinase activity and the ability to be stimulated by insulin. Insulin and poly(L-lysine) also differed in their ability to stimulate the kinase activity of prephosphorylated receptors. Prephosphorylation of the receptors did not affect the stimulation of the kinase by insulin. In contrast, prephosphorylation of receptors resulted in a markedly enhanced ability of poly(L-lysine) to stimulate kinase activity. These studies suggest that the mechanisms by which poly(L-lysine) and insulin activate the kinase are different. In conjunction with other additional evidence, it is suggested that poly(L-lysine) interacts directly with the beta-subunit of the receptor, thereby activating the receptor kinase.

Amino Acid Sequence↗
PubMed1989

The carboxyl terminal segment of the c-Ki-ras 2 gene product mediates insulin-stimulated phosphorylation of calmodulin and stimulates insulin-independent autophosphorylation of the insulin receptor.

Cationic cofactors (e.g., polylysine or histone H2B) are necessary to observe phosphorylation of calmodulin in cell-free systems containing partially purified insulin receptors from a variety of tissues. The highly basic carboxyl terminus of the human c-Ki-ras 2 gene product stimulated both the in vitro phosphorylation of calmodulin and autophosphorylation of the beta-subunit of the insulin receptor, independently of insulin. Addition of insulin increased phosphate incorporation into calmodulin 2.5 fold. The K0.5 for insulin was approximately 5 x 10(-8) M. Maximal phosphorylation occurred at 120 microM c-Ki-ras 2 in the absence of Ca2+ and was inhibited by free Ca2+ concentrations above 0.1 microM. These data suggest the c-Ki-ras 2 gene product, an endogenous membrane protein, may play an important role in the cellular mechanism of insulin action.

Animals↗
PubMed1989

Calmodulin as substrate for insulin-receptor kinase. Phosphorylation by receptors from rat skeletal muscle.

Calmodulin is a substrate for insulin-receptor kinase obtained from rat adipocytes and hepatocytes and human placenta. In this study, we demonstrate that insulin stimulates the phosphorylation of calmodulin via insulin receptors partially purified from rat skeletal muscle. Phosphorylation of calmodulin was maximal in the presence of Mg2+ and insulin and the absence of Ca2+. Free-Ca2+ concentrations greater than 0.1 microM progressively inhibited phosphorylation with almost total inhibition at 200 microM Ca2+. Insulin-stimulated phosphorylation of calmodulin was dose dependent and saturable with half-maximal effect obtained at approximately 5 x 10(-10) M insulin. There was an absolute requirement for certain basic proteins, e.g., polylysine or protamine sulfate, to obtain phosphate incorporation into calmodulin. Polylysine stimulated the phosphorylation of calmodulin independently of insulin, but this was increased up to sixfold by the addition of insulin. Phosphate incorporation into calmodulin increased with increasing concentration of the substrate up to a saturating concentration of 2.4 microM. The Km for calmodulin was approximately 0.2 microM. Up to 0.15 mol of phosphate was incorporated per mole of calmodulin with tyrosine the predominant amino acid phosphorylated. The observations that calmodulin is phosphorylated by insulin-receptor kinase from all three classic target organs for insulin confirm that calmodulin is a general substrate for this kinase and suggest that Ca2+ and calmodulin may be components of the insulin-signaling mechanism.

Amino Acids↗
PubMed1988

Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations.

Insulin stimulates autophosphorylation of the beta subunit of its receptor and activates the associated tyrosine kinase. This kinase, in turn, phosphorylates a number of specific protein substrates; however, the functional and structural identity of these substrates is largely unknown. In this study, we demonstrate that insulin also stimulates the phosphorylation of calmodulin by rat hepatocyte insulin receptors partially purified by wheat germ agglutinin affinity chromatography. Phosphorylation occurred predominantly on tyrosine residues and had an absolute requirement for insulin receptors, divalent cations, and certain basic proteins. Maximal 32P incorporation was observed at an insulin concentration of 5 X 10(-9) M, and the K0.5 for insulin was approximately 4 X 10(-10) M. Phosphorylation of calmodulin was dependent upon ATP, saturating at 100 microM ATP with a K0.5 of 30 microM. Insulin-stimulated phosphorylation of calmodulin was also dependent upon Mg2+ or Mn2+, but was approximately 12-fold greater in the presence of Mg2+. Maximal phosphorylation was observed in the absence of Ca2+ and was inhibited at Ca2+:EGTA ratios greater than 0.8 (0.16 microM free Ca2+). Certain basic proteins, such as polylysine, histone Hf2b, and protamine sulfate, were necessary to observe insulin-stimulated phosphorylation of calmodulin. The relative amount of insulin-stimulated phosphorylation of calmodulin observed in the presence of each of these proteins differed. Maximal insulin-stimulated phosphorylation was observed in the presence of polylysine. These data suggest that both Ca2+ and calmodulin may participate in the early post-receptor events in the cellular mechanism of insulin action in hepatocytes.

Adenosine Triphosphate↗
PubMed1988

Characteristics of calmodulin phosphorylation by the insulin receptor kinase.

Calmodulin is a substrate for the insulin receptor kinase. The time sequence of events resulting in insulin-stimulated phosphorylation of calmodulin was analyzed at a number of different insulin concentrations using partially purified solubilized insulin receptor preparations from rat adipocytes. The respective insulin concentrations needed to reach half-maximal binding, phosphorylation of the beta-subunit of the insulin receptor, and phosphorylation of calmodulin were 4.5 X 10(-10), 4.3 X 10(-10), and 3.9 X 10(-10) M, respectively. At all insulin concentrations, the time to reach 50% of the maximum (defined as the value obtained at 60 min) occurred in the sequence: insulin binding less than beta-subunit phosphorylation less than calmodulin phosphorylation. Insulin binding and beta-subunit phosphorylation occurred almost immediately, whereas there was a lag phase preceding calmodulin phosphorylation. Although stoichiometry was generally low under routine assay conditions (0.01-0.10 mol phosphate/mol calmodulin), it could be increased 4.3 +/- 0.5-fold (n = 5) by pretreating the calmodulin with 0.1 N NaOH. Insulin-stimulated phosphorylation of calmodulin was exclusively on tyrosine residues. The calmodulin molecule in animals contains only two tyrosine residues, located at positions 99 and 138. The amount of phosphate incorporation into a semisynthetic calmodulin (VU1) which contains only one of these tyrosine residues (tyrosine-138) was half that obtained with porcine or chicken calmodulin. Therefore, insulin, via its receptor kinase, stimulates the phosphorylation of calmodulin; calmodulin can be phosphorylated on both tyrosine residues 99 and 138.

Adipose Tissue↗
PubMed1985

Hypophosphataemia in acute pancreatitis.

Serum phosphate levels were measured in a group of 78 patients admitted to hospital with a confirmed diagnosis of acute pancreatitis. Forty-seven (60%) of these patients developed significant hypophosphataemia (less than 0,8 mmol/l) within 4 days of admission. Serum phosphate levels were generally normal on admission, declined on the 2nd-5th days, and returned to normal within 3-14 days. A history of previous excessive alcohol intake was obtained from 42 patients (89%) in the hypophosphataemic group and from 28 (90%) in the normophosphataemic group. In view of the close similarities between the complications of acute pancreatitis and those of hypophosphataemia, including 'toxic psychosis', haematemesis, haematological disturbances, bone infarcts, respiratory failure, abnormal liver function and hyperglycaemia, it is suggested that at least some of these complications could be due to phosphate depletion. Phosphate replacement may be of benefit in their prevention.

Acute Disease↗
PubMedDate not supplied

Bilateral Duane's syndrome.

Two cases with bilateral Duane's Syndrome are presented. Both showed immediate large angle exotropia following bilateral medial rectus recession for esotropia. In both cases, the forced duction test was then found to be positive in adduction and negative after disinsertion of the lateral rectus. It is suggested that both the medial rectus muscles and lateral rectus muscles are tight in bilateral Duane's Syndrome and that seemingly simultaneous recession of the medial and lateral rectus muscles would be indicated.

Adolescent↗