A K+-selective,three-state channel from fragmented sarcoplasmic reticulum of frog leg muscle

Summary Sarcoplasmic reticulum (SR) vesicles from frog leg muscle were fused with a planar phospholipid bilayer by a method described previously for rabbit SR. As a result of the fusion, K⁺-selective conduction channels are inserted into the bilayer. Unlike the two-state rabbit channel, the frog channel displays three states: a nonconducting (closed) state and two conducting states and . In 0.1m K⁺ the single-channel conductances are 50 and 150 pS for and , respectively. The probabilities of appearearance of the three states are voltage-dependent, and transitions between the closed and states proceed through the state. Both open states follow a quantitatively identical selectivity sequence in channel conductance: K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺>Cs⁺. Both open states are blocked by Cs⁺ asymmetrically in a voltage-dependent manner. The zero-voltage dissociation constant for blocking is the same for both open states, but the voltage-dependences of the Cs⁺ block for the two states differ in a way suggesting that the Cs⁺ blocking site is located more deeply inside the membrane in the than in the state.     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

Characteristics of the Inhibitory Effect of Calmodulin on Specific [125I]Omega-Conotoxin GVIA Binding to Crude Membranes from Chick Brain

The characteristics of the inhibitory effect of calcium ion (Ca²⁺)/calmodulin (CaM) on specific [¹²⁵I]-omega-conotoxin GVIA (¹²⁵I--CTX) binding and on the labeling of ¹²⁵I--CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca²⁺/CaM depended on the concentrations of free Ca²⁺ and CaM. The IC₅₀ values for free Ca²⁺ and CaM were about 2.0 × 10^–8 M and 3.0 g protein/ml, respectively. The inhibitory effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290–309), but not by the calcineurin inhibitor FK506. Ca²⁺/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca²⁺ channel ₁ subunits) with ¹²⁵I--CTX using a cross-linker. These results suggest that Ca²⁺/CaM affects specific ¹²⁵I--CTX binding sites, probably N-type Ca²⁺ channel ₁ subunits, in crude membranes from chick whole brain.     

Dipole moments of scorpion toxins direct the interaction towards small- or large-conductance Ca2+-activated K+ channels

Ca²⁺-activated K⁺ channels consist of alarge family of membrane proteins, among which twogroups have been characterized by electrophysiologicalcriteria, the small conductance (SK) and the largeconductance (BK) Ca²⁺-activated K⁺channels. Scorpion toxins that block K⁺ channelsexhibit a common three-dimensional structureconstituted of a short -helix connected bydisulfide bonds to a -sheet. The leiurotoxin I(LTX1) related toxins interact specifically with theSK channel via basic residues of their -helix,while the charybdotoxin (ChTX) family recognizes theBK channel with basic residues of their -sheet.In an attempt to better understand thestructure–activity relationships of these toxins andthe characteristics of the electrostatic interactionswith the receptor site, we investigated theelectrostatic potential supported by natural toxinsand a synthetic analogue to find out if it may help inunderstanding the molecular mechanisms involved inthis peptide–protein interaction.     

Modulation of Na+-channels by neurotoxins produces different effects on [3H]ACh release with mobilization of distinct Ca2+-channels

1. Voltage-gated Na⁺ channels are responsible for initiation and conduction of action potentials. The arrival of an action potential at nerve terminal increases intracellular Na⁺ and Ca²⁺ concentrations. Calcium entry into neurons through voltage-dependent calcium channels is associated with a variety of intracellular processes. Scorpion neurotoxins have been used as tools to investigate mechanisms involved in neurotransmitter release. Tityustoxin (TsTX) is an -type toxin that delays Na⁺-channel inactivation. Toxin- (TiTX-) is a -type toxin that induces Na⁺-channel activation at resting potentials.2. In the present work, we describe the effects of both toxins on [³H]acetylcholine ([³H]ACh) release from rat cerebrocortical synaptosomes, in the presence or absence of the calcium channels blockers: -conotoxin-GVIA (-CgTx), 1 M; -agatoxin-IVA (-Aga), 30 nM; -conotoxin-MVIIC (-MVIIC), 1 M; or verapamil, 1M.3. TsTX evokes [³H]ACh release in a concentration-dependent manner with a gradual increase up to saturation at concentrations of 500 nM. However, release of ACh evoked by TiTX- was not linear regarding the toxin concentration. The [³H]-ACh release evoked by TsTX or TiTX- was partially inhibited by -CgTx or -Aga, and blocked with -MVIIC. Verapamil (1 M) had no effect. Tetrodotoxin blocked [³H]ACh release evoked by both toxins.4. These results show that different actions on Na⁺-channels produce different effects on [³H]ACh release with involvement of distinct presynaptic Ca²⁺-channels, which supports the idea that sodium channels may modulate neurotransmitter release.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

A domain of the α-subunit of rabbit phosphorylase kinase shows homologies with regions of rabbit α-tropomyosin,human EGF receptor,and the α chain of bovine S-100 protein

Sequence comparison of the -subunit of phosphorylase kinase with -tropomyosin revealed 32% identity, and 49% similarity, between the region of -tropomyosin coded by exon 5 and a 39 amino acid segment of the kinase subunit. A subsequence of the -subunit segment and a sequence overlapping the same -subunit region are homologous with: (a) a region of the cytoplasmic domain of EGF receptor (50% identity) and (b) a Ca²⁺-binding domain of the chain of S-100 protei (50% identity) respectively. Statistical analysis shows that these homologies are significant. The biological implication of the above similarities is discussed.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

A solid-phase synthesis of three aza-, iminoaza- and reduced aza-peptides from the same precursor

Using the Boc-strategy, a step-by-step synthesis on the PAM solid supportof three aza-, iminoaza- and reduced aza-peptide homologues is described.From the same hydrazinocarbonyl peptide-PAM precursor, the coupling ofeither a Boc-amino acid or a Boc-amino aldehyde gives rise to an aza-peptideor an iminoaza-peptide containing theC-CO-NH-N-CO-NH-C orC-CH=N-N-CO-NH-C surrogate of the peptide motif, respectively. In situreduction of the latter by NaBH₃CN leads to a reducedaza-peptide containing theC-CH₂-NH-N-CO-NH-C moiety. The key step synthesis of thehydrazinocarbonyl peptide-PAM precursor is carried out by coupling on thegrowing peptide chain the N-Boc-aza-amino acid chloride obtained by theaction of triphosgene on the corresponding N-Boc-hydrazine. Thesemodifications have been introduced in position 1-2 of the YLGYLEQLLRbenzodiazepine-like decapeptide     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

Structural Features of Pregna-D′-pentaranes Determining Their Interaction with Three Rat Proteins

Competition of a number of progesterone 16,17-cycloalkane derivatives with ³H-labeled ligands for the binding sites of rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16,17-cyclopropanoprogesterone, and 16,17-cyclopent-3-enoprogesterone and the selective ligands for serum pentaranophilin are 6-methyl-16,17-cyclohexanopregna-1,4-diene-3,20-dione and 3-hydroxy-16,17-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D we studied decreased the affinity of ligands for all the three proteins. The substitution of the ⁵-3-hydroxy grouping for the ⁴-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3,4-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

Characterization of Na,K-ATPase genes in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) and comparative genomic organization with rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

A combination of molecular and in silico approaches was employed to assemble a survey of Na, K-ATPase genes contained in the ancestrally tetraploid genome of the Atlantic salmon (Salmo salar). Molecular characterization of genomic clones coding for the subunit revealed two single genes (1a and 2) and two pairs of presumably homeologous genes (1b/i-ii and 1c/i-ii). Each of the six genes showed high sequence similarity to isoforms previously isolated from rainbow trout and extensive structural differences relative to putative orthologs in the human genome. In silico analysis of expressed sequence tag (EST) collections indicated that at least five (1a, 1b, 1c, 2, and 3) and four (1a, 1b, 2, and 3b) subunit isoforms are expressed in Atlantic salmon. Meiotic linkage analysis further showed that Na, K-ATPase genes are dispersed throughout the salmon genome, with the exception of two multigene clusters on linkage groups AS-22 and AS-28. Duplicate gene copies for the isoform 1b were assigned to linkage groups with multiple homeologous anchors (AS-22 and AS-23), while 2 duplicates suggested a new homeologous affinity between AS-05 and AS-28. In addition, the comparison of linkage arrangements with rainbow trout also showed that the genomic organization of Na, K-ATPase genes is consistent with the evolutionary conservation of syntenic chromosome regions between these species.Electronic Supplementary Material Supplementary material is available for this article at .     

T-cell repertoire in a strain of transgenic C57BL/6 mice with the HLA-DRA gene on the X-chromosome

We have established a strain of transgenic mice in which the HLA-DRA gene was integrated into the X-chromosome and the xenogeneic mixed isotype molecule, DRE^b, was expressed in a cell type-specific manner, although the transgenic DRA gene contained only 268 base pairs of the 5-flanking region. The DRE^b molecules expressed in the transgenic mice functioned as major histocompatibility complex (MHC) class II to select T-cell repertoire, and to stimulate mixed lymphocyte reaction. In female transgenic mice homozygous for HLA-DRA (DR-B6-F-homo) and male transgenic mice (DR-B6-M), DRE^b molecules were expressed in almost all of the MHC class II A^b-positive cells. In contrast, the expression of DRE^b molecules in female transgenic mice hemizygous for HLA-DRA (DR-B6-F-hemi) was found only in part of the A^b positive cells, and the proportion of cells expressing the DRE^b molecules varied due to random inactivation of one of the X-chromosomes. Clonal deletions of the T cells and mature thymocytes bearing Tcrb-V5 and Tcrb-V11, which are eliminated from the peripheral repertoire in mice expressing self-superantigen and MHC class II E molecules, were incomplete in DR-B6-F-hemi as compared with those in DR-B6-F-homo, and were correlated with the proportion of DRE^b-positive spleen cells. These observations suggested that the number of bone marrow-derived cells expressing DRE^b molecules was critical for clonal deletions of Tcrb-V5⁺ and Tcrb-V11⁺ T cells in the thymus.     

Interleukin-2-dependent long-term cultures of low-density lymphocytes allow the proliferation of lymphokine-activated killer cells with natural killer,Tiγ/δ or TNK phenotype

Summary We have developed a culture system for longterm growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum antitumor cytotoxicity. The system allows the exponential growth of monocyte-depleted low-density lymphocytes in the presence of human serum and recombinant human interleukin-2 (10³ U/ml), alone or in combination with interleukin-1 or (both at 10 U/ml). Eighteen cultures were established from 18 normal adult donors. The membrane phenotypes of the final LAK cell population, assessed by a panel of monoclonal antibodies (mAb), consist of three main types: (a) NKH-1⁺, Ti/^–, Ti/^–, and CD3^– lymphocytes; (b) NKH-1⁺, Ti/^–, Ti/⁺, and CD3⁺ lymphocytes and (c) NKH-1⁺, Ti/⁺, Ti/^– and CD3⁺ lymphocytes. Northern blot analysis showed that all these cell populations express relatively high levels of perforin RNA, particularly cells exhibiting the first phenotype. This culture system may provide a tool for cellular and molecular studies on the mechanisms of antitumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced cancer.     

Molecular modeling of the rabbit colonic (HKα2a) H<Superscript>+</Superscript>, K<Superscript>+</Superscript> ATPase

A model of the HK2a subunit of the rabbit colonic H⁺, K⁺ ATPase has been generated using the crystal structure of the Ca⁺² ATPase as a template. A pairwise sequence alignment of the deduced primary sequences of the two proteins demonstrated that they share 29% amino acid sequence identity and 47% similarity. Using O (version 7) the model of HK2a was constructed by interactively mutating, deleting, and inserting the amino acids that differed between the pairwise sequence alignment of the Ca⁺² ATPase and HK2a. Insertions and deletions in the HK2a sequence occur in apparent extra-membraneous loop regions. The HK2a model was energy minimized and globally refined to a level comparable to that of the Ca⁺² ATPase structure using CNS. The charge distribution over the surface of HK2a was evaluated in GRASP and possible secondary structure elements of HK2a were visualized in BOBSCRIPT. Conservation and placement of residues that may be involved in ouabain binding by the H⁺, K⁺ ATPase were considered and a putative location for the subunit was postulated within the structure.Figure Possible architecture of the HK2a subunit. The residue in green is the lysine (position 517, Fig. 1) that lies in the nucleotide binding pocket and the residue in red is the aspartic acid at the phosphorylation site (position 385). Based on an alignment with the Ca⁺² ATPase, ten transmembrane helices were modeled into HK2a. The ten transmembrane helices are drawn as rods and shown in different colors for clarity. From left to right, the transmembrane helix designations are M10 (blue), M7 (gray), M8 (purple), M9 (orange), M5 (pink), M6 (green), M3 (brown), M4 (cyan), M2 (teal), and M1 (almond).     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney α-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification

Summary NADH inhibition of bovine kidney -ketoglutarate dehydrogenase complex was compared at 10 m free Ca²⁺ or in the absence of Ca²⁺ (i.e., < 1.0 nM free Ca²⁺). In the presence of Ca^2–, NADH inhibition was appreciably decreased for a wide range of NADH : NAD⁺ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 m free Ca²⁺ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca²⁺ on the S_0.5 for -ketoglutarate which was decreased by Ca²⁺ with a half-maximal effect at a similar concentration. The effect of Ca²⁺ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the -ketoglutarate dehydrogenase complex is required to elicit the effect of Ca²⁺ on NADH inhibition.At a fixed -ketoglutarate concentration (50 m), removal of Ca² reduced the activity of the -ketoglutarate dehydrogenase complex by 8,5-fold (due to an increase in S_0.5 for -ketoglutarate) and, in the presence of different NADH : NAD⁺ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxP_i) or a combination of the phosphate potential and NADH :NAD⁺ ratio are also described. The possibility that the level of intramitochondrial free Ca²⁺ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.     

Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca²⁺ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC were included for comparison.The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C , ₁, , , and / were all present in rat midbrain cytosolic extract, PKC , ₁, , and / were present in spleen cytosol, and PKC and / were present in COS 7 cell cytosol. The predominance of and activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay.The PKC activity assay was configured such that the Ca²⁺ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca²⁺-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca²⁺, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca²⁺ whereas in the presence of Ca²⁺, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca²⁺. Maximum values for Ca²⁺-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca²⁺-dependent PKC isoform activity in a Ca²⁺ free environment. The greater potency of mezerein on PKC was confirmed using PKC and further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca²⁺ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca²⁺-dependent isoforms in Ca²⁺ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca²⁺. The concentration-dependence of DOG was examined on PKC and PKC further purified from brain by HAP chromatography, revealing that DOG was equally potent on both of these isoforms derived from brain and on recombinant PKC . However, [³H]PDBu binding data using PKC purified from several sources gave very different IC₅₀ values when DOG was used as a displacer, and in general these values correlated with the EC₅₀ values recorded from the activity assay.The data presented here indicate that there are distinct differences in the activator pharmacology of different native PKC isoforms and between the same isoform expressed in different tissues, either because of post-translational modifications or some other tissue specific factor.