Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca²⁺ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca²⁺. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca²⁺ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca²⁺ flux via the protein kinase autophosphorylation mechanism.Abbreviations SDS sodium dodecyl sulfate - EGTA ethylene glycol bis (-aminoethyl ether) - N,N,N,N tetra acetic acid - EDTA ethylenediamine-tetraacetic acid - cAMP cyclic adenosine 35 monophosphate This work is supported by grants from the Medical Research Council of Canada (JHW), the Heart and Stroke Foundation of Alberta (JHW and RKS) and the Heart and Stroke Foundation of Saskatchewan (RKS)     

Differential distribution of calpain in human lymphoid cells

Calpain, a calcium-activated neutral proteinase, is ubiquitously present in human tissues. To determine if lymphoid cells implicated in pathogenesis of demyelination may harbor calpain in a functionally active form, we determined both Calpain and mCalpain activities in human lymphoid cell lines. DEAE-cellulose and phenylsepharose column chromatography were used to isolate the enzyme from the natural inhibitor, calpastatin. Lymphocytic lines (CCRF-CEM, MOLT-3, MOLT-4, M.R.) showed predominance of Calpain (55–80%) whereas the monocytic line (U-937) showed prodominance of mCalpain (77%). Proportion and subcellular distribution of both isoforms varied among cell lines. Calpains isolated from U-937 cells degraded myelin basic protein. These results indicate that human lymphoid cells harbor functionally active calpain that can degrade myelin components in vitro. The study suggests a degradative role for calpain in demyelinating diseases.     

Mammalian myristoyl CoA: protein N-myristoyltransferase

Myristoyl CoA:Protein N-myristoyltransferase (NMT) is the enzyme which catalyses the covalent transfer of myristate from myristoyl CoA to the amino-terminal glycine residue of protein substrates. Although NMT is ubiquitous in eukaryotic cells, the enzyme levels and cellular distribution vary among tissues. In this article, we describe the properties of mammalian NMT(s) with reference to subcellular distribution, molecular weights, substrate specificity and the possible involvement of NMT in pathological processes. The cytosolic fraction of bovine brain contains multiple forms of NMT activity whereas bovine spleen contains only a single form. In bovine brain and spleen, the cytosol contained majority of NMT activity. In contrast, rabbit colon and rat liver NMT activity was predominantly particulate. Regional differences in NMT activity have been observed in both rabbit intestine and bovine brain. Results from our laboratory along with the existing knowledge, provide evidence for the existence of tissue specific isozymes of NMT.     

Influence of carbon and nitrogen sources on glutathione catabolic enzymes inCandida albicans during dimorphism

The effect of carbon sources, glucose and sucrose, and nitrogen sources such as ammonia, glutamate andl-citrulline on the activities of glutathione metabolic enzymes has been studied. Yeast and mycelial cells were used to identify changes in activity levels of glutathione reductase (GSSGR), glutathione transferase (GST), glutathione peroxidase (GPX) and -glutamyl transpeptidase (GGT). Enzyme activities from cells grown in sucrose media were lower than in glucose media regardless of the enzyme tested, morphological form, or the growth interval. In all enzymes except GST, activity was higher in yeast form than in mycelia, regardless of nitrogen source, with lower activity from 24 to 72 h than at 96 h. In citrulline media, yeast form showed the maximum GST, GGT, and GPX activity. In ammonia-amended media, mycelia showed maximum activity in GGT, whereas in glutamate media, mycelia showed the maximum activity in GST. Also, the type of nitrogen source had no effect on GPX activity in the mycelial form. Finally, changing the nitrogen source showed no significant effect on GSSGR activity, either in the yeast or mycelial form.     

Heterogeneity in photosystem I. Evidence from cation-induced decrease in Photosystem I electron transport

The rate of electron transfer through Photosystem I (reduced 2,6-dichlorophenol indophenol (DCIPH₂ → methylviologen) in a low-salt thylakoid suspension is inhibited by Mg²⁺ both under light-limited and the light-saturated conditions, the magnitude of inhibition being the same. The 2,6-dichlorophenol indophenol (DCIP) concentration dependence of the light-saturated rate in the presence and in the absence of Mg²⁺ shows that the overall rate constant of the photoreaction is not altered by Mg²⁺. With N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethylphenylenediamine as electron donor only the light-limited rate, not the light-saturated rate, is inhibited by Mg²⁺ and the magnitude of inhibition is the same as with DCIP as donor. The results are interpreted in terms of heterogeneous Photosystem I, consisting of two types, PS I-A and PS I-B, where PS I-A is involved in cation-regulation of excitation energy distribution and becomes unavailable for DCIPH₂ → methyl viologen photoelectron transfer in the presence of Mg²⁺.     

Positive cooperativity in the adherence between elementary bodies of Chlamydia trachomatis strain UW-31 and HeLa cells

Abstract The adherence of purified elementary bodies of Chlamydia trachomatis strain UW-31 to monolayer cultures of HeLa 229 cells exhibited kinetic evidence of positive cooperativity. An abrupt increase in the rate of adherence occurred as chlamydial dose was increased. Only freshly isolated chlamydiae showed this behavior. In the presence of the lectin wheat germ agglutinin, the stimulated adherence showed typical Michaelis-Menten kinetics. The results suggest that chlamydiae may promote their own binding to the host-cell surface, and the lectin, when cell-bound, may provide additional chlamydiae-binding sites.     

Relationship between P700 turnover (m second) and NADP reduction as a function of ferredoxin concentration in isolated broken chloroplasts

Steady-state electron flux through P₇₀₀ (t $\text{1}\text{2}$ 20 msec) and concomitant rate of NADP reduction have been measured under weak actinic illumination as a function of concentration of ferredoxin added to broken chloroplasts isolated from peas. At suboptimal concentrations of ferredoxin this P₇₀₀ is not sufficient to account for the NADP reduction. At high concentrations ferredoxin inhibits the rate of NADP reduction without affecting the P₇₀₀ flux under short wavelength illumination. Under far red illumination P₇₀₀ flux is also inhibited by ferredoxin at high concentrations. Addition of 5 mM Mg⁺⁺ increases the rate of NADP reduction at all concentrations of ferredoxin under both kinds of illumination, while P₇₀₀ flux is inhibited under short wavelength illumination and remains unchanged under far red illumination. The results indicate that the observed (20 msec) P₇₀₀ is not involved in NADP reduction.     

Measurement of nitrite reductase in leaf tissue of Vigna mungo

The enzyme nitrite reductase (EC 1.6.6.4) is generally assayed in terms of disappearance of nitrite from the assay medium. We describe a technique which allowed estimation of the enzyme level in leaf tissues of Vigna mungo (L). Hepper in terms of the release of the product (NH₃) of the enzyme reaction. The technique is offered as an alternative, possibly more convenient method for assay of nitrite reductase in plant tissue in vivo.     

Depression of mitogen response in spleen cells from reticuloendotheliosis virus-infected chickens and their suppressive effect on normal lymphocyte response

Spleen cells and peripheral blood lymphocytes from chickens infected with reticuloendotheliosis virus (REV) were depressed in their responsiveness to phytohemagglutinin (PHA). When spleen cells from uninfected chickens were co-cultured with spleen cells from chickens infected with REV at 2 weeks of age, the PHA response by the normal cells was completely suppressed. Although spleen cells from chickens infected with REV at 6 or 9 weeks of age were also suppressed in their ability to respond to PHA, they did not suppress the mitogenic response of normal cells in mixed cultures.