Human creatine kinase: isolation and sequence analysis of cDNA clones for the B subunit, development of subunit specific probes and determination of gene copy number

cDNA clones for human B creatine kinase were isolated from human brain and placenta libraries. The entire coding and 3' untranslated regions, as well as 23 bp of the 5' untranslated region were sequenced. Complete sequence identity was found among the clones, with the exception of an area of heterogeneity among the 3' untranslated region of the brain and placenta clones. A 77.7% nucleotide sequence identity was found between the coding region of human B creatine kinase and our previously reported human M creatine kinase. In contrast, no homology was found in the 3' untranslated regions. Probes were constructed from the nonconserved 3' untranslated regions of human M and B creatine kinase and were shown to be highly specific. Southern transfers of total genomic DNA derived from human placenta and digested to completion with several restriction enzymes were probed with the MCK and BCK specific probes producing single hybridization bands. These results suggest that creatine kinase M and B are single copy genes in the human genome.     

Creatine ethyl ester: a new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K(m) and V(max) values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

Direct assay for creatine kinase by reversed-phase high-performance liquid chromatography

A direct assay for creatine kinase (CK) activity was developed based on the separation and quantitation of adenosine triphosphate (ATP) by high-performance liquid chromatography. The total incubation time is 13 min and the elution time for ATP is 16 min. Using lyophilized CK as the sample, a sensitivity in the range of 8 U/l (units/liter) was obtained. The method presented also has clinical significance in that CK levels in serum can easily be determined with minimal sample preparation. Using serum samples from a healthy patient and a heart attack victim, activities of 26.6 U/l and 609.0 U/l, respectively, were obtained. Because of the direct measurement of ATP, this method eliminates the coupled reactions encountered in the common spectrophotometric and colorimetric methods of analysis resulting in a simpler and inexpensive assay.     

A novel approach for evaluating tyrosine kinase activity based on the radioimmunological determination of phosphotyrosine.

A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide.     

Direct determination of creatine kinase equilibrium constants with creatine or cyclocreatine substrate

The equilibrium constants of two reactions catalyzed by rabbit muscle creatine kinase with creatine or cyclocreatine as substrate were determined by 31P-NMR. The value of the equilibrium constant with creatine as substrate was 172.10(7) M(-1) in agreement with previous work (Veech, R.L., Lawson, J.W.R., Cornell, W. and Krebs, H.A. (1979) J. Biol. Chem 254, 6538-6547). The value with cyclocreatine was 5.62.10(7) M(-1) and the ratio of the two constants is 30.6. It was possible to determine the ratio of the two equilibrium constants in a reaction mixture containing both substrates since it was found that the 31P resonances of P-creatine and P-cyclocreatine were well resolved. The ratio of K1/K2 determined in such experiments was 34.6, of the same order as previously reported by Annesley and Walker (Annesley, T.M. and Walker, J.B. (1977) Biochem. Biophys. Res. Commun. 74, 185-190).     

A complete system for identifying inhibitors of creatine kinase B

We have developed a complete system for discovery of lead compounds as inhibitors of creatine kinase B. In this article, we describe production and purification of the recombinant protein, conditions and features of an optimized high-throughput screening assay, and results of our implementation of the system using a diverse compound library.     

A new colorimetric method for determination of creatine phosphokinase

A new colorimetric method has been developed for the determination of creatine phosphokinase (CPK). This method is based on the reaction of creatine, formed enzymatically from creatine phosphate and ADP, with p-nitrophenylglyoxal (PNPG) under alkaline conditions to produce a colored product which absorbs maximally at 480 nm. At 25 degrees C the reaction was complete after 10 min in 0.1 M sodium pyrophosphate, pH 12, containing 0.15 M sodium ascorbate. The colored product was stable for at least 24 h and obeyed Beer's law in the range 0.005-0.05 mM creatine. The color reaction was used to determine the activity of CPK in serum and tissue extracts. The results obtained by this method agreed well with the results obtained by other available methods for CPK determination. However, the PNPG method was more rapid and more sensitive than other colorimetric methods and required a single chromogenic reagent.     

A microchip-based enzyme assay for protein kinase A.

A microchip-based enzyme assay for protein kinase A is described. The microchips were prepared by standard photolithographic techniques. The assay reagents were placed in wells on the microchips, and electroosmosis was used to transport aliquots of these reagents into the network of etched channels, where the enzymatic reaction takes place. Protein kinase A catalyzes the transfer of a phosphate group from ATP to the serine residue of the heptapeptide LeuArgArgAlaSerLeuGly (Kemptide). The outcome of the enzymatic reaction was assessed by performing an on-chip electrophoretic separation of the fluorescently labeled peptide substrate and product. All liquid-handling steps were performed by controlling the electroosmotically driven flow from reagent and buffer wells using electrical current. On-chip dilutions of the peptide substrate, ATP and H-89, a known protein kinase A inhibitor, were performed and the kinetic constants (K(m), K(i)) of these compounds were determined. This prototype assay demonstrates the usefulness of the microchips for performing enzymatic assays for which fluorogenic substrates cannot easily be designed.     

Structure and expression of the human creatine kinase B gene

Various cDNAs for creatine kinase type B (CK-B) were isolated from human cDNA libraries using a 26-oligonucleotide guess-mer probe. One of the cDNAs appeared to be almost full-length and contained an open reading frame coding for the 381 amino acid residues of the human CK-B polypeptide. The nucleotide sequences of the translated region as well as the primary protein structure show a high degree of homology with known CK-B and CK-M sequences of other vertebrates. The level of CK-B RNA as a measure of CK-B gene activity was determined in various human tissues and cultured cells. Our results confirm that CK-B is expressed in a tissue-specific manner and give support to the previously proposed relation between CK-B gene activity and cell proliferation. Screening of genomic DNA with various cDNA regions as probes revealed that there is only one CK-B gene per haploid genome. Gene cloning and sequencing indicated that CK-B is coded for by a relatively small gene of 3.2 kb in size, which is partially overlapped by an HTF island (A. P. Bird (1986) Nature (London) 321, 557-558) with an extremely high G + C content at its 5' end.     

Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B

Background: The Amplicor™ HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates.Objective: The performance of the Amplicor™ HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor™ HBV Monoitor assay was compared to the Digene Hybrid Capture™ System HBV DNA assay for the quantitation of HBV in patient sera.Study design: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays.Results: The detection limit was found to be 10³ copies/ml with the Amplicor™ PCR assay compared to 10⁶ to 10⁷ copies/ml with the Digene™ hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor™ PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10³ and 10⁷ copies/ml and all of them tested below the detection limit with the hybridization assay.Conclusion: The Amplicor™ HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.     

Creatine ethyl ester: A new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K _m and V _max values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

A new assay for the determination of phosphoribosylamine.

This report describes a new assay for glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) (EC 2.4.2.14) based on the formation of a stable compound between the ribose-5-phosphate moiety of phosphoribosylamine and [³⁵S]cysteine. The assay is comparable in sensitivity to the [¹⁴C]glutamine assay, and its potential in the evaluation of NH₃ utilization by this enzyme is documented. The stoichiometry of the enzymatic reaction and the analysis of the ribose-5-phosphate/cysteine compound demonstrate that 1 mol of phosphoribosylamine (or ribose-5-phosphate) combines with 1 mol of [³⁵S] cysteine to form this compound. Preliminary studies with a number of carbohydrates indicate that an aldopentose phosphate is most reactive with cysteine. The substitution of a phosphate, pyrophosphate, or purine ring at C-1 of the pentose markedly reduces its reactivity with cysteine.     

Detection of carriers for duchenne muscular dystrophy. Quality control of creatine kinase assay

Summary A report by Bullock and coworkers has emphasized the need for standardization of the CK assay in carrier detection for DMD. A collaborative study, according to a well-specified design and involving two laboratories in Paris and Lyon, indicates that the reliability of this assay can be improved provided that special attention is paid to information about participating subjects, laboratory protocol, and repeated sampling. On a random sample of young women, mean and variance of 1n(CK) are 1.66 and 0.015 respectively. Within and between variance components are in a 3:4 ratio, homogeneous between populations. Additional use of a common test serum should insure good reliability of this assay among laboratories.     

Purification of mitochondrial creatine kinase: Two interconvertible forms of the active enzyme

The purification of creatine kinase from beef heart mitochondria is described. The purified enzyme appears as a single band after electrophoresis on SDS gels. Electrophoresis on cellulose acetate followed by staining for creatine kinase activity shows two forms of the enzyme. The slower migrating (m-1) form upon concentration is converted to the more rapidly migrating form (m-2). The reverse conversion occurs if the m-2 is incubated with β-mercaptoethanol. These results are consistent with a reversible oxidation of protein sulfhydryl group (s).     

Microbiological assay for quantitative determination of polyoxin B

Polyoxin B, an antifungal agro-antibiotic, is used for treating serious plant diseases. Until now there have been no reports on the antibiotic quantification in liquid in spite of its need for fermentation studies. This work reported a microbiological assay, the cylinder-plate method, for the determination of polyoxin B. The results of assay were treated statistically by analysis of variance (ANOVA) and were found linear in the range of 10–500 μg/ml (r² = 0.998), precise (intra-assay: relative standard deviation (R.S.D.) = 0.64; inter-assay: R.S.D. = 1.70) and accurate. This newly established method was applied to determine the antibiotic titer in Streptomyces cacaoi fermentation with comparison to HPLC analysis. The microbiological assay was satisfactory for polyoxin B quantification, and a simple, sensitive, cost-effective and specific agar diffusion bioassay for polyoxin B was thus developed. This work also demonstrated the usefulness of the microbiological assay for quantitative analysis of antibiotics in fermentation.     

Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate

Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 mM Hepes or phosphate buffer, pH 7.5, 100 microM PK-1, 100 microM [gamma-32P]ATP, 3 mM MgCl2, 12 mM KCl, and 20-200 ng of catalytic subunit assayed at 30 degrees C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.