Recombinant expression and purification of the bovine acidic Seminal Fluid Protein

The acidic Seminal Fluid Protein (aSFP), a 12.9 kDa protein is a maker for bovine semen freezability possibly due to its antioxidant activity and effect on sperm mitochondrial function. However, its precise function on sperm preservation during freezing thaw is poorly understood. The use of recombinant DNA technology allows new approaches on the study of function and structure of proteins, and its production in procaryote systems offers several advantages. The present work describes the recombinant expression of the bovine aSFP and its binding properties. A cDNA library from the bovine seminal vesicle was used as template for amplification of the aSFP coding region. The amplicon was cloned into a pET23a (+) vector and transformed into E.coli BL21 pLysS strain. The recombinant expression was obtained in E coli. One step ion immobilized affinity chromatography was performed, resulting in high yield of purified protein. To determine the bioactivity of the r aSFP, the protein was incubated in different concentrations with 10 7 spermtozoa at 37°C for 5 h. Western blotting and fluorescence microscopy analyses showed the ability of the recombinant aSFP to attach to the spermatozoa. Based on our results, the described method can be used to obtain mg levels of recombinant aSFP.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ¡Ý36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women.

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ≥36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

Troponin I: an update on clinical utility and method standardization.

Cardiac troponin I (cTnI) is now widely recognized as one of the preeminent biochemical markers for the diagnosis of myocardial injury. The biochemical specificity of this biomolecule for cardiac tissue has forced a reevaluation of the diagnostic criteria for non-Q-wave acute myocardial infarction, unstable angina, acute coronary artery disease, and minor myocardial injury. Further, its use by clinicians has revolutionized the way that chronic and acute heart diseases are both diagnosed and managed. Unfortunately, the standardization of cardiac troponin I assays is problematic. Up to 20-fold variation of serum cTnI mass determinations may be observed for a given patient sample when measured by different assay systems. As a result, significant ambiguity often exists in the clinical interpretation of serum cTnI concentrations. Recent efforts have been directed toward the biochemical standardization of cTnI assays. However, the heterogeneous nature and biochemical complexity of the serum forms of cTnI and differences of the epitope recognition by the various methods have hindered the harmonization of serum cTnI assays.

Antibodies against a neuron-like (HTB-10 neuroblastoma) cell in children with Tourette syndrome.

BACKGROUND: Similar to the model for Sydenham's chorea, antineuronal antibodies (ANAb), which develop in response to a preceding streptococcal infection, have been speculated to have a role in the development of Tourette syndrome (TS). METHODS: Serum antibodies against the neuron-like HTB-10 neuroblastoma cell were assayed by ELISA methods and Western blot analysis on 41 children with TS (mean age 11.3 years) and 39 control subjects (mean age 12.1 years). RESULTS: Group comparisons of ELISA assay optical density (OD) showed that mean OD values for serum antibodies were not different [control (mean +/- SEM), .506 +/- .076; and TS, .584 +/- .053 (p = .38)]. In contrast, median values [.353 in control subjects and .477 in TS subjects (p = .012)] were significantly different. Western blots identified numerous bands in all TS and control sera with no difference in identified HTB-10 antigens. There was no relationship between the presence of ANAb and age of tic onset, family history, tic severity, attention deficit hyperactivity disorder, or obsessive compulsive disorder. No relationship existed between positive strep titers (ASO > or = 166 and/or antiDNAaseB > or = 170) and ANAb determinations or the severity of tics. CONCLUSIONS: Children with TS have higher median, but not mean, levels of ANAb, as measured by the HTB-10 neuroblastoma cell membrane assay. This assay system identified antibodies in both control and clinical groups and failed to identify a relationship between antibodies and clinical phenotype or one-time markers for streptococcal infection. Further studies are required to define a possible immune-mediated hypothesis for TS.

A Plasmodium vivax vaccine candidate displays limited allele polymorphism, which does not restrict recognition by antibodies.

BACKGROUND: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite. MATERIALS AND METHODS: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP1(19) described thus far. RESULTS: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein. CONCLUSIONS: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.

Monoclonal antibodies, antigens and molecular diagnostics: a practical overview.

The development of monoclonal antibody-based immunochemical assays to measure antibodies, antigens and small molecules, such as drugs, ushered in a revolution in modern diagnostic medicine. The impact of monoclonal antibodies on diagnostic imaging technologies and therapeutic regimes was equally dramatic. Additionally, the application of immunological techniques to the biochemical research lab was partially responsible for the exponential growth in our understanding of human physiology, biochemistry and genetics. Monoclonal antibody technology is limited, however, by the manner in which antigen-antibody interactions can be controlled and by the ability to consistently produce antibodies with appropriate affinity and specificity. Recent advances in recombinant antigen preparation and antibody engineering have been used to enhance the applications of this technology. In this review, current strategies to generate and engineer monoclonal antibodies as well as their clinical applications are summarized. A brief review of molecular diagnostics and its future trend is also included.

Degradation of cardiac troponin I in serum complicates comparisons of cardiac troponin I assays.

BACKGROUND: Up to a 20-fold variation in serum cardiac troponin I (cTnI) concentration may be observed for a given patient sample with different analytical methods. Because more limited variation is seen for control materials and for purified cTnI, we explored the possibility that cTnI was present in altered forms in serum. METHODS: We used four recombinantly engineered cTnI fragments to study the regions of cTnI recognized by the Stratus(R), Opus(R), and ACCESS(R) immunoassays. The stability of these regions in serum was analyzed with Western blot. RESULTS: The measurement of several control materials and different forms of purified cTnI using selected commercial assays demonstrated five- to ninefold variation. Both the Stratus and Opus assays recognized the N-terminal portion (NTP) of cTnI, whereas the ACCESS assay recognized the C-terminal portion (CTP) of cTnI. Incubation of recombinant cTnI in normal human serum produced a marked decrease in cTnI concentration as determined with the ACCESS, but not the Stratus, immunoassay. Western blot analysis of the same samples using cTnI NTP- and CTP-specific antibodies demonstrated preferential degradation of the CTP of cTnI. CONCLUSIONS: The availability of serum cTnI epitopes is markedly affected by the extent of ligand degradation. The N-terminal half of the cTnI molecule was found to be the most stable region in human serum. Differential degradation of cTnI is a key factor in assay-to-assay variation.

The beta subunit of eukaryotic translation initiation factor 2 binds mRNA through the lysine repeats and a region comprising the C2-C2 motif.

Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNAMet have been identified. All such mutations in the beta subunit of eIF2 (eIF2beta) mapped to a region containing a putative zinc finger structure of the C2-C2 type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2beta that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2beta, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C2-C2 motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2beta, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNAMet binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.

Antibodies against human putamen in children with Tourette syndrome.

BACKGROUND: Similar to the model for Sydenham's chorea, antineuronal antibodies, which develop in response to a preceding streptococcal infection, have been speculated to have a role in the development of Tourette syndrome (TS). METHODS: Serum antibodies against human caudate, putamen, and globus pallidus (interna and externa) were assayed by enzyme-linked immunosorbent assay (ELISA) and Western blot techniques and results were correlated with clinical characteristics and markers of streptococcal infection. SUBJECTS: A total of 41 children with TS (mean age, 11.3 years) and 39 controls (mean age, 12.1 years) were included. RESULTS: Compared with controls, TS subjects had a significant increase in the mean (p=0.006) and median (p=0.002) ELISA optical density (OD) levels of serum antibodies against putamen, but not caudate or globus pallidus. Western blots on 20 control and 20 TS serum samples showed that specific antibodies to caudate/putamen occurred more frequently in TS subjects at 83, 67, and 60 kDa; antigens were present in a synaptosomal fraction. TS subjects with a positive family history of tics had higher OD values (p < or = 0.04), but no association was shown with age of tic onset, tic severity, sudden onset of tics, or presence of attention-deficit hyperactivity disorder or obsessive-compulsive disorder. Risk ratio calculations in TS and control groups and in study subjects dichotomized for high and low putamen OD values were similar for titers of antistreptolysin O > or = 166 or antideoxyribonuclease B > or = 170. A subgroup analysis limited to subjects with elevated streptococcal titers, however, showed a significantly (p < or = 0.004) larger number of TS subjects with elevated OD levels. CONCLUSION: Children and adolescents with TS had significantly higher serum levels of antineuronal antibodies against putamen than did controls, but their relation to clinical characteristics and markers for streptococcal infection remains equivocal.

Thrombus Precursor Protein and the measurement of thrombosis in patients with acute chest pain syndrome.

BACKGROUND: The major limitation of current diagnostic methods for the rapid diagnosis of an acute myocardial infarction (AMI) involves the measurement of the effects of ischemic or necrotic processes on myocardial function rather than the detection of the precipitating thrombotic event. The utility of an assay was investigated for thrombus precursor protein (TpP) in the diagnosis of 115 patients with symptoms consistent with myocardial ischemia or infarction of less than six hours in duration. Samples from patients were drawn at 0, 1, 2, 4, 8, 16, and 24 hours post presentation, and creatine kinase, CK-MB, myoglobin, troponin I, and TpP concentrations were determined. Significantly abnormal concentrations of TpP were observed in 15 of 17 patients presenting with an AMI within 6 hours of the onset of symptoms (p < 0.00001), 2 of 8 patients presenting with an AMI after 6 hours from the onset of symptoms (p = 0.0077), 22 of 35 patients with unstable angina (p = 0.00035), 15 of 30 patients with angina (p = 0.00024), 3 of 5 patients with atrial fibrillation (P = 0.00005), 6 of 9 patients with congestive heart failure (p = 0.00002), and 6 of 11 patients with non-cardiac chest pain (p = 0.00139). Non-cardiac chest pain patients with gastroenteritis, esophageal spasm, duodenal ulcer, cocaine overdose, and pericarditis presented with abnormal plasma concentrations of thrombus precursor protein. Peak concentrations of TpP in patients with AMI preceded those of the other markers by two to four hours.

An immunoassay for anti-neuronal antibodies associated with involuntary repetitive movement disorders.

Tourette's syndrome (TS) is a complex neurobehavioral disorder emerging in childhood and is characterized by motor and vocal tics of at least one year in duration. In a portion of patients with TS, environmental (non-genetic) factors may either have an etiologic role or act to modulate the phenotype. One possible environmental factor may be antibodies to central nervous system cells, as sera from several children diagnosed with either TS or Sydenham's chorea contained anti-neuronal antibodies. Using enriched membrane preparations isolated from HTB-10 neuroblastoma cells, a sensitive and specific assay was developed for the determination of human anti-neuronal antibodies associated with involuntary repetitive movement disorders. This assay exhibited between-run and within-run precision of 11.3 percent and 5.9 percent, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of this assay for the diagnosis of TS and TS or chorea are 79.1 percent, 61.2 percent, 61.6 percent, 78.8 percent, and 71.1 percent, 60.9 percent, 68.6 percent, and 63.6 percent, respectively. In addition, there was a significant difference (p < 0.0001) between the mean optical density in the patients with TS and children determined to be clinically "normal".

Comparative sensitivities and specificities of the mass measurements of CK-MB2, CK-MB, and myoglobin for diagnosing acute myocardial infarction.

We evaluated the clinical utility of the mass measurement of the tissue isoform of creatine kinase MB isoenzyme (CK-MB2) in the diagnosis of an acute myocardial infarction (AMI) by determining its sensitivity, specificity, and predictive value relative to those of CK-MB mass and myoglobin. Samples were obtained at 0, 4, 8, and 16 h postpresentation from 100 patients (41% with AMI). The order of sensitivity for the sample proportions taken at 0-2 h from the onset of symptoms was myoglobin > CK-MB2 > CK-MB. At all other time points, the sensitivity of CK-MB2 either equaled or surpassed that of both CK-MB and myoglobin, although the 95% confidence intervals for the population proportions each of these markers overlapped. Of the 41 AMI patients, 31 (76%) exhibited concurrent abnormal increases of CK-MB and %CK-MB2; the other 10 (24%; 8 non-Q wave, 2 Q wave) exhibited abnormal values for %CK-MB2 before their CK-MB exceeded the upper limit of normal. The specificity of myoglobin was statistically lower than that for either CK-MB2 or CK-MB at all time points.

Family history and premature coronary heart disease.

BACKGROUND: We were interested in studying whether a family history of coronary heart disease (CHD) persisted as a significant risk factor for premature coronary heart disease after adjusting for traditional and nontraditional risk factors. METHODS: Ninety-five case patients with documented premature CHD (occurring in a person less than 60 years old and with greater than 50 percent occlusion of a major epicardial vessel or a documented myocardial infarction) and 95 community-based control patients were examined for risk factors including family history, hypertension, diabetes mellitus, sedentary lifestyle, smoking, body mass index, total cholesterol, high-density lipoprotein cholesterol, triglycerides, low-density lipoprotein cholesterol, lipoprotein(a), homocysteine, and fibrinogen. RESULTS: The risk of premature CHD for a positive family history ranged from an odds ratio (OR) of 3.25 for a standard family history of CHD in a first-degree relative, 5.9 for family history of early CHD in a first-degree relative before the age of 45 years, and 6.1 for a strong family history of CHD defined as CHD in at least two first-degree relatives. Family history persisted as a significant risk factor for premature CHD (OR = 3.9, 95 percent confidence interval [CI] 1.8-8.7) in multiple variable models that included traditional and nontraditional risk factors. It was rare, however, for a person with a positive family history not to have at least two other traditional or nontraditional risk factors. CONCLUSIONS: Family history of CHD should not be considered a simple binary risk factor for premature CHD, and a positive family history of CHD indicates that a person is at high risk for premature CHD independent of traditional and nontraditional risk factors.

The effect of high-dose ascorbate supplementation on plasma lipoprotein(a) levels in patients with premature coronary heart disease.

STUDY OBJECTIVE: To determine the efficacy of high-dose ascorbate supplementation in lowering lipoprotein(a) [Lp(a)] levels in patients with premature coronary heart disease (CHD). DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Outpatient clinic. PATIENTS: Forty-four patients with documented premature CHD, defined as confirmed myocardial infarction and/or angiographically determined stenosis of 50% or greater in at least one major coronary artery before age 60 years. INTERVENTIONS: Patients were block randomized on the basis of age, gender, and screening Lp(a) concentrations to receive ascorbate 4.5 g/day or placebo for 12 weeks. MEASUREMENTS AND MAIN RESULTS: High-dose ascorbate was well tolerated and produced a marked elevation in mean plasma ascorbate levels (+1.2 mg/dl; p < 0.001). Multiple linear regression analysis revealed no significant effect of supplementation on postintervention Lp(a) levels (p = 0.39) in a model that included treatment group assignment, and baseline Lp(a) levels. CONCLUSIONS: Our findings do not support a clinically important lowering effect of high-dose ascorbate on plasma Lp(a) in patients with premature CHD.

An immunochemical mass assay for the direct measurement of creatine kinase MB2.

The isoforms of CK-MB have recently received attention as potential biochemical markers for the early diagnosis of an acute myocardial infarction. A sensitive (analytical sensitivity = 0.2 ng/ml) immunochemical mass assay for the direct measurement of the CK-MB2 isoform has been developed by us. This assay utilizes a specific monoclonal capture antibody directed against the B-subunit of CK-MB and a specific monoclonal antibody conjugate directed against the CK-M + lysine subunit. Owing to the lack of a World Health Organization standard for CK-MB, the percent CK-MB2 values had to be normalized by determining both CK-MB and CK-MB2 in assays which differ only in the specificity of the anti-CK-M conjugate. Thus, a related CK-MB immunochemical mass assay utilizing the identical capture antibody and a specific monoclonal antibody conjugate directed against the CK-M subunit was also developed. Analytical sensitivities of the CK-MB and CK-MB2 assays were determined to be 0.5 ng/ml and 0.2 ng/ml, respectively. Both CK-MB and CK-MB2 levels were determined in 46 hospitalized non-AMI patients and 35 non-hospitalized normal patients. Of the 46 hospitalized non-AMI patients (mean age = 70), 26 percent had either CK-MB and/or CK-MB2 values below the level of sensitivity of the CK-MB or CK-MB2 isoform assays. For patients with CK-MB values between 0.5 ng/ml and 2.99 ng/ml, the percent CK-MB2 values ranged from 35 percent to 97 percent. For patients with CK-MB values between 3.0 ng/ml and 6.5 ng/ml, the percent CK-MB2 values ranged from 23 percent to 72 percent. Similar results were obtained for the non-hospitalized group. This assay appears to be useful in determining the clinical utility of CK-MB2.

Comparison of three immunoassays for the determination of N-acetylprocainamide. Determination of false elevations by a polyclonal antibody-based method.

The performance of the ROCHE FP NAPA reagent, a newly reformulated monoclonal antibody-based fluorescence polarization immunoassay (FPIA) for N-acetylprocainamide, was compared with two commercially available polyclonal antibody-based immunoassays, the Abbott TDx and Syva EMIT. Although excellent correlation was observed between the ROCHE FP NAPA reagent and the Syva EMIT reagent, a positive bias was observed in the correlation between the ROCHE FP NAPA reagent and the Abbott TDx reagent. The bias in the TDx method was investigated further by the concurrent high-performance liquid chromatography (HPLC) analysis of patient samples. Insignificant differences (p < 0.05) were observed between the HPLC and ROCHE FP values. The TDx system, however, yielded significantly higher (p < 0.0005) results than did HPLC.

[Preparation and use of fish fillet infusion as a basic medium for culturing bacteria]. / Preparo e utilização do infuso de filé de pescado como meio básico para o cultivo de bactérias.

The authors present the first results on the utilization of fish infusion (IFP) as a basic medium for the cultivation of bacteria. The infusion was obtained from a common marine fish, corvina (Micropogonias furnieri) according to the technique used in the preparation of beef infusion broth. Streptococcus pyogenes, S. pneumoniae, Neisseria meningitidis, Campylobacter jejuni, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis were cultured in liquid and solid media prepared with IFP as well as in recommended standard media. Solid media used for cultivation of S. pyogenes, S. pneumoniae, N. meningitidis and C. jejuni were supplemented with 5% of defibrinated sheep blood and for the latter, substances recommended to increase aerotolerance were included in solid and liquid media. All of these strains grew on the media prepared with IFP except S. pneumoniae when cultured in IFP diluted 1:2 with a sodium chloride solution. Only S. pyogenes produced colonies smaller than those of the standard medium. No more differences were detect in the observation of colony morphology. The growth of E. coli, K. pneumoniae, S. marcescens, P. aeruginosa, S. aureus and B. subtilis was measured in liquid media after 8 hours. In solid media, the growth index was expressed by dividing the number of colonies produced in IFP-agar and Nutriente Agar by the number of colonies on Trypticase soy agar plates. Some differences were observed in colonial size and morphology when compared with those generated in standard media. The average value obtained from the analyses of total proteins by biuret reaction in twelve batches of IFP was 5.03 mg/ml. The experiments showed that culture media prepared with IFP supported the growth of bacterial strains used in this work. It suggests that fish infusion has promising conditions of being an alternative substrate for cultural purposes.

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.

The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase.

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.