Sets of peptides with defined sequences, each on a separate spot, were synthesized simultaneously on continuous cellulose membranes (SPOTs membranes), which were originally designed for epitope studies. The applicability of the membrane-bound peptides as substrates for protein kinases was tested using protein kinase A, protein kinase C and casein kinases I and II as model enzymes. We found that the peptide-membrane complexes can serve as kinase substrates. Our results suggest that membrane-bound peptides offer a new potential for the investigation of substrate specificity of protein kinases. An advantage to this method is that there is no need for substrate identification and separation, which is required with high-volume random peptide libraries. Membrane-bound peptides may even form a basis for kinase assays with peptides lacking multiple basic amino acids, required for separation of the substrates in conventional assays. Problems connected with protein kinase substrate specificity can be investigated in any laboratory using the rapid and inexpensive SPOTs technique, as neither costly apparatus nor special experience in peptide synthesis is necessary.
Like many other large cities, Stockholm is facing increased urbanization with densification of infrastructure as a result. At the same time, implementation of the Baltic Sea Action Plan and the EU Water Framework Directive is expected to result in more stringent effluent quality demands. The current situation gives rise to new challenges for the municipal wastewater treatment plants (WWTPs). This paper describes how two of Sweden's largest municipal water organizations; Stockholm Vatten and Syvab, will face these challenges using ultrafiltration (UF) membrane bioreactor (MBR) technology. The effluent requirements for the rehabilitated plants are expected to be tightened to 6 mg/l and 0.2 mg/l for total nitrogen (TN) and total phosphorus (TP), respectively.
The possible applicability of ceramic waste as partial replacement of Portland cement was studied. For this purpose, two ceramic wastes and two Portland cements of different countries (Argentine and Czech Republic) were analysed. After characterization of the materials used (chemical and mineralogical composition and specific surface), the effect of ceramic waste replacement (8, 16, 24, 32 and 40 % by mass) was analyzed. The pozzolanic activity, the heat released rate and the hydration products were determined at 2, 7 and 28 days. Results show that ceramic wastes have pozzolanic activity with both portland cements. At early age, the dilution effect governs the properties and finally the pozzolanic reaction improves the performance of blended cements.
The influence of inactivation of protein kinase C on the kinetics of the catalytic phosphorylation of stereoisomeric peptides Lys-Arg-Pro-Ser-Glu-Arg-Ala Lys-Tyr, where the L-amino acids were successively replaced by the corresponding D-isomers, was analysed by means of an integrated rate equation.It was shown that the completeness of peptide phosphorylation in these reactions was governed by the ratio of the rates of substrate phosphorylation and enzyme inactivation.As the rate of the phosphorylation of the substrates depended remarkably on the position of the D-amino acid in the peptide structure, the yields of the phosphorylated products were rather different.Besides that the degree of substrate conversion was also dependent on the enzyme concentration in the reaction mixture, which also determined the rate of catalysis.
Protein kinases are enzymes that catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The specificity of selected model protein kinases was studied at three different levels using a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. A new class of protein kinase bi-substrate-analogue inhibitors was designed on the basis of adenosine-5’-carboxylic acid derivatives, where a short arginine containing peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. These compounds showed high inhibitory potential against two basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity towards the acidophilic kinases CK1 and CK2. The inhibitors were efficiently applied for affinity purification of PKA using MgATP as well as L-arginine as eluting agents. Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings and its substrate specificity was studied using a set of synthetic peptides. These were derived from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR (where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine) was defined from a study using arrays of systematically varied peptides attached to cellulose membrane (SPOTsTM membranes). The SuSy2 derived peptides were also found to be efficient substrates for mammalian PKC, but showed low reactivity in the case of PKA. On the basis of this peptide motif, a positionally oriented peptide library approach based on ESI-MS detection of phosphopeptides in initial velocity conditions was designed for quantitative kinetic characterization of protein kinase specificity profiles. On the basis of the obtained data an optimal peptide substrate for PKC, FRRRRSFRRR, was designed. The specificity of protein kinase A was studied using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and comparison of the obtained data with the data from previous studies on structurally altered peptide substrates revealed that amino acid alterations in short peptide substrates cause stronger effects on the phosphorylation rate than the corresponding alterations in the protein substrate L-PK.
Several proteins in the mammalian endoplasmic reticulum are substrates for protein kinases. Many unidentified phosphoproteins from this compartment are described in the literature, and this prompted us to try to identify at least the more dominant ones. When solubilized bovine and murine microsomes were phosphorylated with protein kinase CK2 and [32P] ATP and separated on SDS-PAGE, the corresponding autoradiogram showed three dominant 32P-labeled proteins. These three [32P]phosphoproteins were identified as calcium-binding proteins (CaBP) 1,2, and 4 after purification on a MonoQ column followed by SDS-PAGE, proteolytic cleavage and subsequent amino acid sequencing of the purified 32P-labeled peptides. All three were also phosphorylated by an endogenous kinase, found by us to be of the CK2 type. This kinase phosphorylated CaBP1 N-terminally at serine 427. Of the three proteins, only CaBP4 was previously known to be a substrate of CK2. The newly identified substrates CaBP 1 and 2 are members of the thioredoxin family and have a signal tetrapeptide in the C-terminal of the protein for retention in the ER. Serines and/or threonines in the C-terminal were phosphorylated in CaBP1 when the endogenous CK2 was used as protein kinase. A protein with the same molecular mass as CaBP1 on SDS-PAGE was phosphorylated when intact hepatocytes were grown in the presence of [32P] phosphate. The in vitro phosphorylation with protein kinase CK2 can be used as a specific and sensitive method for identification of CaBP1, 2, and 4 in microsomes
