Isolation of the light-harvesting chlorophyll a/b--protein complex from thylakoid membranes of barley by adsorption chromatography on controlled-pore glass.

The light-harvesting chlorophyll $\text{a}\text{b}$-protein complex has been isolated from barley thylakoids by a rapid, single-step procedure involving adsorption chromatography on controlled-pore glass columns. The Triton X-100-solubilized complex contains a polypeptide of apparent molecular weight, 26,000; the 0.25% Triton X-100 light-harvesting chlorophyll $\text{a}\text{b}$-protein has spectral characteristics consistent with its assumed in vivo state. On the same column free chlorophyll and carotenoids have been separated from chlorophyll-protein complex 1, but this complex contained many polypeptides other than those associated with chlorophyll. This method is potentially suitable for the isolation of other thylakoid membrane proteins. It may also be generally applicable for fractionation of intrinsic membrane proteins from other sources and for separation of mixed Triton X-100-lipid micelles.     

Depolarisation-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Factors Determining the Magnitude of the Response

Abstract: The sequence of molecular events linking depolarisation-dependent calcium influx to the release of neurotransmitters from nerve terminals is unknown; however, calcium-stimulated protein phosphorylation may play a role. In this study the incorporation of phosphate into proteins was investigated using an intact postmitochondrial pellet isolated from rat cerebral cortex. The rate and relative incorporation of label into individual phosphoproteins depended on the prelabelling time and buffer concentrations of calcium and phosphate. After prelabelling for 45 min, depolarisation caused a >20% increase in the labelling of 10 phosphoproteins, and this initial increase was maximal with 41 mM K⁺ for 5 s, or 30 μ M veratridine for 15 s, in the presence of 1 mM calcium. Both agents also led to an initial dephosphorylation of four phosphoproteins. Depolarisation for 5 min led to a significant decrease in the labelling of all phosphoproteins. All of the depolarisation-stimulated changes in protein phosphorylation were calcium-dependent. The depolarisation conditions found to optimally alter the phosphorylation of synaptosomal proteins find many parallels in studies on calcium uptake and neurotransmitter release. However, the uniform responses of such a large number of phosphoproteins to the multitude of depolarisation conditions studied suggest that the changes could equally well relate to recovery events such as biosynthesis of neurotransmitters and regulation of intraterminal metabolic activity.     

The conversion of ATP to IMP by muscle surface enzymes

These experiments showed that an isolated muscle bundle could be used to study, simultaneously, ion transport and the activity of surface enzymes. Frog muscles were carefully dissected and incubated for four hours in Ringer's solution containing a tris buffer and 3 mM ATP; at various times samples of the medium were chromatographed and analysed spectrophotometrically for nucleotide content. Samples of the final medium were analysed for inorganic phoshpate and for ammonia. The results demonstrated the conversion of adenosine triphosphate to inosine monophosphate, via adenosine diphosphate and adenosine monophosphate. Under the conditions of the experiment IMP was detected on chromatograms within 20 minutes of incubation; at the end of four hours the media had concentrations of 2.3 mM IMP, 4.4 mM inorganic phosphate and 2.6 mM ammonia, showing a stoichiometric relation among the products formed. There was convincing evidence that the enzymes involved (ATPase, adenylate kinase and AMP deaminase) must be situated close to or on the muscle surface. No effect of ouabain (1 μM) on the activity of the ATPase, adenylate kinase or deaminase could be found in these experiments, but the drug inhibited Na and K recovery from a Na-loaded, K-depleted state.     

Tyrosine Hydroxylase Phosphorylation in Bovine Adrenal Chromaffin Cells: The Role of Intracellular Ca2+ in the Histamine H1 Receptor-Stimulated Phosphorylation of Ser8, Ser19, Ser31, and Ser40

Abstract: Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H₁ receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca²⁺. In contrast, the H₁-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 µ M ) and significantly reduced by prior exposure to caffeine (10 m M for 10 min) to deplete intracellular Ca²⁺. Trypticphosphopeptide analysis by HPLC revealed that the H₁ response in the presence or absence of extracellular Ca²⁺ resulted in a major increase in the phosphorylation of Ser¹⁹ with smaller increases in that of Ser⁴⁰ and Ser³¹. In contrast, although a brief stimulation with nicotine (30 µ M for 60 s) also resulted in a major increase in Ser¹⁹ phosphorylation, this response was abolished in the absence of extracellular Ca²⁺. These data indicate that the mobilization of intracellular Ca²⁺ plays a crucial role in supporting H₁-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.     

A role for calcium/calmodulin kinase(s) in the regulation of GABA exocytosis

A possible role for protein kinases in the regulation of GABA exocytosis in nerve endings was investigated. The effect on the release of the radioactive neurotransmitter ([³H]GABA) from mouse brain synaptosomes of several protein kinase inhibitors was estimated after treatment with 37 mM K⁺ in the absence of external Na⁺, a condition under which [³H]GABA release is completely Ca²⁺ dependent. Among the inhibitors one group inhibit the kinases by binding to the catalytic site (i.e. staurosporine and H7) and others (TFP, sphingosine and W7) act on the regulatory site of protein kinases. The compounds of the second group, which are reported to inhibit calmodulin dependent events and the increase in cytosolic Ca²⁺ (Ca _i ) induced by high K⁺ depolarization, were the most efficient inhibitors of [³H]GABA release. The selective inhibitor of CaMPK II, KN-62, also markedly diminished [³H]GABA release as well as the increase in Ca _i induced by high K⁺. The kinase inhibitors from the first group that are unable to diminish the increase in Ca _i induced by high K⁺ were also less efficient inhibitors of [³H]GABA release even at high concentrations. The present results indicate that at the doses tested all the drugs inhibit to some extent the release of the Ca²⁺ dependent fraction of [³H]GABA perhaps by inhibiting a CaMPK II mediated phosphorylation step triggered by depolarization and facilitated by the elevation of Ca _i . In addition, the second group of antagonists and KN-62 inhibit the elevation of Ca _i to high K⁺ thus exhibiting a higher efficiency on [³H]GABA release than the first group of antagonists.     

Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Partition of substrates and protein kinase activities with triton X-100.

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase activity catalysing the phosphorylation of endogenous substrates. Extraction of membrane fragments with Triton X-100 solubilized less than 20% of the kinase activity and left the major part of the endogenous substrates in the insoluble fraction.     

Ascorbic acid and copper in linoleate oxidation. 3. Catalysts in combination

In promoting oxidation of 0.02 m potassium linoleate in a buffered (pH 7.0) aqueous dispersion at 37 degrees C, ascorbic acid at low concentrations (1.8 x 10(-6) and 1.8 x 10(-5) m) in combination with copper (1.3 x 10(-7) to 1.3 x 10(-3) m) had greater catalytic activity than the additive activity of the two catalysts individually. Possible explanations for the enhanced catalysis include reduction of copper by ascorbic acid to the cuprous form, increased concentration of semidehydroascorbic acid radical, and formation of a metal-ascorbic acid-oxygen complex. Some combinations of ascorbic acid (1.8 x 10(-4) and 1.8 x 10(-3) m) and copper (1.3 x 10(-6) and 1.3 x 10(-3) m) inhibited the formation of conjugated dienes but not the oxidation of ascorbic acid, and caused rapid loss of part of the conjugated dienes that were already present. It is suggested that free-radical inhibitors formed by the combination of catalysts inhibit initiation of lipid oxidation but not copper-catalyzed oxidation of ascorbic acid. Effects of the inhibitory combinations on changes in UV absorption by conjugated dienes, and absorbance in the TBA test, indicate the presence of at least two conjugated dienes that differ in stability.     

Phosphorylation of myelin basic protein by an adenosine 3′:5′-cyclic monophosphate-dependent protein kinase (Short Communication)

Myelin basic protein was shown to be a substrate for protein kinase from rabbit muscle. One of the major sites of phosphorylation was the serine residue in the sequence Gly-Arg-Gly-Leu-Ser-Leu. The arginine residue in this sequence is known to be a substrate for a protein methylase.     

Circulating T-cell response to Helicobacter pylori infection in chronic gastritis

Background. Helicobacter pylori elicits a specific humoral and cellular immune response. There is increasing evidence that the type of T-cell response contributes to clinical outcome in H. pylori infection.
Materials and Methods. The host response to H. pylori infection in 34 subjects with chronic gastritis was examined in terms of T-cell proliferation and cytokine production in whole-blood cultures stimulated or unstimulated with H. pylori acid-glycine extract antigens (AGE).
Results. The proliferative response in whole-blood cultures was similar for both H. pylori –positive and –negative subjects stimulated with H. pylori AGE. While an increase in interferon-γ (IFN-γ) production was observed from both H. pylori –positive and –negative subjects with gastritis, significantly higher levels of IFN-γ were detected in the former when stimulated with H. pylori AGE. In contrast, interleukin 4 (IL-4) was undetectable regardless of antigen stimulation. However, if an in situ IL-4 antibody capture assay was used, antigen-independent production of IL-4 was detected, but there was no difference between H. pylori –positive and –negative subjects with gastritis. After eradication of H. pylori , antigen-induced production of IL-4 was increased, with no decrease in the levels of secretion of IFN-γ. IL-4 production was dependent on CD4+ T cells, as addition of anti-CD4 but not anti-CD8 mouse monoclonal antibody or matched IgG isotype to the whole-blood culture inhibited the production of IL-4.
Conclusion. The results suggest that a shift toward a balanced Th1-Th2 response due to an increase in antigen-induced IL-4 production from CD4+ T cells follows eradication. We suggest that the downregulation of mucosal inflammation consequent on reduction in antigen levels or removal of downregulation after eradication of H. pylori contributes to this shift in cytokine balance.