Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[α-P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[α-³²P]ADP in the dark with a K_d value of 8 μM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[α-³²P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[α-³²P]ADP, both the ADP/ATP carrier and the β subunit of the membrane-bound F₁-ATPase are covalently labeled. The binding specificity of 2-azido-[α-³²P]ADP for the β subunit of F₁-ATPase is ascertained by prevention of photolabeling of isolated F₁ by preincubation with an excess of ADP.     

The recognition of two specific binding sites of the adenine nucleotide translocase by palmitoyl CoA in bovine heart mitochondria and submitochondrial particles.

Palmitoyl CoA which is an effective inhibitor of adenine nucleotide transport is able to remove bound [¹⁴C]ADP and [³H]atractylate from the translocator on the outer side of the inner mitochondrial membrane. Bongkrekic acid, when added to the incubation medium prior to palmitoyl CoA, can prevent the removal of bound [¹⁴C]ADP from the membrane by palmitoyl CoA, however, bongkrekic acid is ineffective if palmitoyl CoA is added first. Upon incubation with inverted submitochondrial particles, both palmitoyl CoA and bongkrekic acid prevent the uptake and transport of [¹⁴C]ADP by the particles. Moreover, when the submitochondrial particles are preincubated with [¹⁴C]ADP, palmitoyl CoA, like bongkrekic acid, is unable to remove the bound nucleotide from the inner face of the carrier. Thus, palmitoyl CoA which has a high affinity for the translocator on both sides of the inner mitochondrial membrane, nevertheless, interacts differently with the carrier on each side of the membrane. This suggests that the translocase contains binding sites in two specific states both of which accommodate palmitoyl CoA.     

Thioesterase and protein deacylase activities of porcine pancreatic phospholipase A2

The thioesterase activity of porcine pancreatic phospholipase A₂ has been investigated with non-phospholipid substrates. The acyl-CoA hydrolase activity towards acyl-CoA derivatives is specific for long chain fatty acids (14 C, 16 C) but is unable to hydrolyze short chain acyl-CoA compounds (below 8 C). The same enzyme also shows protein deacylase activity liberating [³H]palmitic acid from [³H]palmitoyl-acyl carrier protein.     

Evidence for the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into dolichol derivatives and glycoproteins by calf brain membranes

Incubating white matter membranes with UDP-N-acetyl-[¹⁴C]glucosamine in the presence of Mg²⁺ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl₃CH₃OHH₂O (10:10:3) extract after incubating with only UDP-N-acetyl-[¹⁴C] glucosamine, Mg²⁺, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[¹⁴C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl₃CH₃OHH₂O (10:10:3). The [GlcNAc-¹⁴C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [mannose-¹⁴C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[¹⁴C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[¹⁴C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β-N-acetylglucosaminidase, indicating that all of the N-acetyl-[¹⁴C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.     

Targeting of auxin carriers to the plasma membrane: differential effects of brefeldin A on the traffic of auxin uptake and efflux carriers

Monensin and brefeldin A (BFA), inhibitors of Golgi-mediated protein secretion, rapidly perturb the transport catalytic activity of specific plasma membrane-associated efflux carriers for indole-3-acetic acid (IAA) and inhibit polar transport of IAA. To determine if these responses result solely from perturbation of the efflux carrier or whether specific auxin uptake carrier function is also affected, the influence of BFA on the cellular transport of a range of auxins with contrasting affinities for specific auxin uptake and efflux carriers was investigated in zucchini (Cucurbita pepo L.) hypocotyl tissue. In-flight addition of BFA (3 · 10⁻⁵ mol · dm⁻³) caused a rapid (lag < 10 min) and substantial (fourfold) increase in the rate of [1-¹⁴C]IAA net uptake by zucchini hypocotyl tissue. In the presence of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA; 3 · 10⁻⁶ mol · dm⁻³), BFA slightly reduced the rate of [1-¹⁴C]IAA net uptake. Stimulation of [1-¹⁴C]IAA net uptake by BFA was concentration-dependent. In the absence of BFA, net uptake of [1-¹⁴C]IAA exhibited the characteristic biphasic response to increasing concentrations of competing cold IAA but in the presence of BFA, [1-¹⁴C]IAA uptake decreased smoothly with increase in concentration of competing unlabelled IAA, indicating a loss of auxin efflux carrier activity but retention of functional uptake carriers. The half-time for mediated efflux of [1-¹⁴C]IAA from preloaded zucchini tissue was substantially increased by BFA (t_1/2 = 51 min, controls; 107 min, BFA-treated). Treatment with BFA and/or NPA did not significantly affect the net uptake by, or efflux from, zucchini tissue of [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([1-¹⁴C]2,4-D), a substrate for the auxin uptake carrier but not the auxin efflux carrier. Uptake of [1-¹⁴C]2,4-D declined smoothly with increasing concentrations of competing unlabelled IAA whether or not BFA was included in the uptake medium, confirming the failure of BFA to perturb auxin uptake carrier function. Transport of 1-[4-³H]naphthaleneacetic acid (1-NAA) exhibited little response to BFA or NPA, confirming that it is only a weakly transported substrate for the efflux carrier in zucchini cells. Received: 12 November 1997 / Accepted: 27 January 1998     

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.

The synthesis of ¹⁴C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-¹⁴C]guanine or [8-¹⁴C]hypoxanthine, but not from [8-¹⁴C]adenine, was demonstrated in a cell-free extract from N₂-fixing nodules of cowpea (Walp.). The ¹⁴C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the ¹⁴C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.     

Adenine nucleotide translocation in plant mitochondria

The rapid translocation of external ADP-[^14C]by corn mitochondria is inhibited by high concentrations of atractyloside with enhanced inhibition occurring in the presence of Mg²⁺. This translocation is also inhibited by AMP or ATP but CDP, GDP, IDP or UDP have little effect. Backward exchange of internal ADP-[¹⁴C] occurs in the presence of AMP, ADP or ATP but is not promoted by other nucleoside diphosphates. It is suggested that the adenine nucleotide (AdN) carrier is specific for ADP and ATP and that apparent translocation of AMP is a result of adenylate kinase activity. The translocated ADP can be separated into 3 components: (1) atractyloside-insensitive binding; (2) carrier-bound ADP saturated at ca 30 μM external ADP; and (3) exchanged ADP saturated as ca 5 μM external ADP. It is suggested that the adenine nucleotide carrier of plant mitochondria possesses similar properties to the classical carrier of vertebrate mitochondria.     

Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

ADP- and epinephrine-elicited release of [3H]guanylylimododiphosphate from platelet membranes: Implications for receptor-Ni stoichiometry

Epinephrine-promoted release of [³H]guanylylimidodiphosphate ([³H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha₂-adrenergic recpetors and N_i, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [³H]Gpp(NH). The amount of [³H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [³H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N_i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N_i.     

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [¹⁴C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF₁. [¹⁴C]ADP binding exhibits a monophasic concentration curve with a K_m of 2 μM.2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF₁ conformation is established.3. Re-binding of [¹⁴C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF₁, [¹⁴C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF₁ is converted to a non-exchangeable form.4. Energy-dependent adenine nucleotide exchange on CF₁ is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.     

An 18 000-dalton protein metabolically labeled by polyamines in various mammalian cell lines

The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [¹⁴C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoreses. Under such incubation conditions, a single protein band with an apparent molecular weight of 18 000 was radioactively labeled. [¹⁴C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of γ-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [¹⁴C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation.     

A Novel Method for Chemical Modification of Functional Groups Other Than a Carboxyl Group in Proteins byN-Ethyl-5-phenylisooxazolium-3′-sulfonate (Woodward's Reagent-K): Inhibition of ADP-Induced Platelet Responses Involves Covalent Modification of Aggregin, an ADP Receptor

The chemical reaction ofN-ethyl-5-phenylisooxazolium-3′-sulfonate (Woodward's Reagent-K, WR-K) with a carboxyl group yields an enol ester that cannot be reduced by sodium borohydride in an aqueous solution, while other nucleophiles such as sulfhydryl, hydroxyl, amino, and imidazole groups, react with WR-K to yield unsaturated ketones that are capable of being reduced by sodium borohydride in an aqueous medium. Aggregin, a 100-kDa protein on the surface of human blood platelets has been identified as an ADP receptor. Autoradiography of the gels obtained by sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the samples of solubilized human blood platelets modified by WR-K and then reduced by tritiated sodium borohydride (NaB[³H]₄) showed the presence of a prominent band corresponding to a 100-kDa radiolabeled protein. Labeling of platelets by WR-K and NaB[³H]₄was inhibited by ADP, ATP, and thiol group modifying reagents. WR-K blocked completely labeling of platelets by [β-³²P]-8-(4-bromo-2,3-dioxo-butylthio)adenosine-5′-diphosphate, an ADP-affinity analog that selectively and covalently labels aggregin (Puri, R. N., Kumar, A., Chen, H., Colman, R. F., and Colman, R. W. (1995)J. Biol. Chem.256, 24482–24488). WR-K also inhibited ADP-induced platelet shape change, aggregation, and mobilization of intracellular Ca²⁺and blocked ADP-induced inhibition of stimulated adenylate cyclase activity. The results show conclusively that WR-K inhibited ADP-induced platelet responses by preventing binding of ADP to aggregin and suggest that ADP binding domain of aggregin contains an essential thiol group. The method of labeling proteins by WR-K and NaB[³H]₄, hitherto not used to distinguish among functional groups modified by WR-K, offers a useful and convenient alternative to previously used ultraviolet spectral methods which cannot be used to investigate the modified proteins in intact cellular systems.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [¹²⁵I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([¹²⁵I]-GN₈-AzPEA) and a [¹²⁵I]-labeled 2- (4-aminophenyl)ethylamino conjugate ([¹²⁵]-GN₈-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligasaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)₈ showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)₈, cellopentaose nor α-1,4 linked N-acetylgalactosamine octamer (GalNAc)₈ at concentrations as high as 25 μM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the ‘high-affinity binding site’ previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Transglutaminase activity in pancreatic islets

1. Pancreatic islet homogenates catalyze, in a Ca²⁺-dependent fashion, the incorporation of [2,5-³H]histamine, [1,4-¹⁴C]putrescine, [1,2-³H]agmatine, [¹⁴C]methylamine, L-[U-¹⁴C]lysine in N,N-dimethylcasein. 2. Using [2,5-³H]histamine as the amine donor, the K_m for Ca²⁺ and histamine amounts to 90μM and 0.7 mM, respectively. 3. The incorporation of [2,5-³H]histamine into N,N-dimethylcasein is inhibited by monodansylcadaverine, N-p-tosyl glycine, bacitracin and methylamine, the relative extent of inhibition depending on the respective concentrations of Ca²⁺, inhibitor and amine donor. 4. Bacitracin and methylamine, but not N-p-tosyl glycine, cause a dose-related inhibition of glucose-stimulated insulin release. 5. It is concluded that, in pancreatic islets, the Ca²⁺-responsive transglutaminase activity plays a critical role in the process of glucose-induced insulin release.     

A study on the exchange of tightly bound nucleotides on the membrane-associated chloroplast ATP synthase complex

Light-induced exchange of tightly bound ADP on the membrane-associated chloroplast coupling factor 1 (CF₁) was concluded to be a two-step mechanism involving a loose enzyme-ADP complex (Strotmann, H., Bickel-Sandkötter, S. and Shoshan, V. (1979) FEBS Lett. 101, 316–320). Rapid binding of [¹⁴C]ADP to the coupling factor after deenergization of thylakoids which were illuminated in the presence of [¹⁴C]ADP was suggested to reflect the conversion of loosely bound to tightly bound ADP. Experimental data of the present paper support the assumption of an intermediate enzyme form with loosely bound ADP: (a) the amplitude of the rapid binding phase is independent on the concentration of uncoupler added in the light; (b) the amplitude is virtually unaffected by dilution of the medium [¹⁴]CADP concentration; (c) high concentrations of unlabeled ADP are required to reduce the rapid binding phase while binding of medium [¹⁴C]ADP is inhibited by unlabeled ADP in the micromolar range. These results exclude the possibility that the rapid initial formation of tightly bound [¹⁴C]ADP on deenergization might be caused by an energized nucleotide-free enzyme form which is able to pick up [¹⁴C]ADP from the medium at a higher rate than the deenergized nucleotide-free form. At saturating [¹⁴C]ADP concentrations in the light, the amount of the loose enzyme-ADP complex is about 35%, while 65% of the coupling factors contain a tightly bound ADP. Dissociation of the loose complex is slow in the absence of medium nucleotides but accelerated if ADP is present, suggesting that ADP binding to another site of the enzyme promotes release of the former ADP molecule. The significance of the loosely bound nucleotide in the catalytic mechanism is discussed.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F₁ with Mg-ATP was required for the binding of the natural inhibitor, IF₁, to F₁ to form the inactive F₁-IF₁ complex. When F₁ was incubated in the presence of [¹⁴C]ATP and MgCl₂, about 2 mol ¹⁴C-labeled adenine nucleotides were found to bind per mol of F₁; the bound ¹⁴C-labeled nucleotides consisted of [¹⁴C]ADP arising from [¹⁴C]ATP hydrolysis and [¹⁴C]ATP. The ¹⁴C-labeled nucleotide binding was not prevented by IF₁. These data are in agreement with the idea that the formation of the F₁-IF₁ complex requires an appropriate conformation of F₁. (2) The ¹⁴C-labeled adenine nucleotides bound to F₁ following preincubation of F₁ with Mg-[¹⁴C]ATP could be exchanged with added [³H]ADP or [³H]ATP. No exchange occurred between added [³H]ADP or [³H]ATP and the ¹⁴C-labeled adenine nucleotides bound to the F₁-IF₁ complex. These data suggest that the conformation of F₁ in the isolated F₁-IF₁ complex is further modified in such a way that the bound ¹⁴C-labeled nucleotides are no longer available for exchange. (3) ³²P_i was able to bind to isolated F₁ with a stoichiometry of about 1 mol of P_i per mol of F₁ (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of ³²P_i to the F₁-IF₁ complex. Thus, not only the nucleotides sites, but also the P_i site, are masked from interaction with external ligands in the isolated F₁-IF₁ complex.