EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and P_i release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.     

Aminoacyl RNA domain of turnip yellow mosaic virus Val-RNA interacting with elongation factor Tu

Turnip yellow mosaic virus (TYMV) Val-RNA forms a complex with the peptide elongation factor Tu (EF-Tu) in the presence of GTP: the Val-RNA is protected by EF-Tu·GTP from non-enzymatic deacylation and nuclease digestion. The determination of the length of the shortest TYMV Val-RNA fragment that binds EF-Tu·GTP leads us to conclude that the valylated aminoacyl RNA domain equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T arm is sufficient for complex formation. Since the aminoacyl RNA domain is also sufficient for adenylation by the ATP(CTP):tRNA nucleotidyltransferase, an analogy can be drawn between these two tRNA-specific proteins.     

Conformational activation of the EF-Tu·GTPase activity Stimulation of the 2′(3′)-O-l-phenylalanyladenosine-promoted EF-Tu·GTPase upon binding of the tRNA lacking the terminal adenosine residue

The elongation factor Tu (EF-Tu) dependent GTPase (in the presence of aurodox) is stimulated by analogs of the aminoacyl tRNA 3′-terminus in the following order: A-Phe < C-A-Phe < C-C-A-Phe. The GTPase-promoting activity of A-Phe is strongly enhanced by tRNA-C-C (devoid of 3′-terminal adenosine residue) but not by intact tRNA-C-C-A. On the other hand, the activity of C-A-Phe as the EF-Tu·GTPase promoter is only slightly enhanced by tRNA-C-C.     

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNA^Gly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNA^Gly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these ‘non-proteogenic’ tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of ‘proteogenic’ aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNA^Gly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.     

X-ray diffraction data for (1→3)-α-d-glucan triacetate

The crystal structures of (1→3)-α-d-glucan triacetates were studied by X-ray diffraction measurements on fibre diagrams. The oriented films annealed in water at high temperature were of higher crystallinity and occurred as two crystalline polymorphs (GTA I and GTA II) depending on the samples and also the annealing temperature. All reflections in GTA I were indexed with a pseudo-orthorhombic unit cell with a = 1·753, b = 3·018 and c(fibre axis) = 1·205 nm. From the fibre repeat data coupled with the density data and the presence of only the (003) reflection on the meridian, an extended three-fold helical structure was proposed. Although some reflections in GTA II split from the layer lines, the basic unit cell was a monoclinic system with a = 1·685, b = 3·878, c (fibre axis) = 1·210 nm and γ = 112·2°. A similar three-fold structure to GTA I was proposed from the almost identical fibre repeat and the conformational analysis on (1→3)-α-d-glucan. It was concluded that, on acetylation, the d-glucan structure changed from the fully extended two-fold helix to the extended three-fold accompanied by some extent of chain shrinking.     

Synthesis and characterization of homochiral polymeric S-malato molybdate(VI): toward the potentially stereospecific formation and absolute configuration of iron-molybdenum cofactor in nitrogenase

Reaction of sodium or potassium molybdate and excess malic acid in a wide range of pH values (pH 4.0–7.0) resulted in the isolation of two cis-dioxo-bis(malato)-Mo(VI) complexes, viz. Na₃[MoO₂H(S-mal)₂] and K₃[MoO₂H(S-mal)₂]·H₂O (H₃mal=malic acid). The sodium complex is also characterized by an X-ray structure analysis, showing that the mononuclear Mo units are linked together via very strong symmetric CO₂···H··· O₂C-hydrogen bond [2.432(5) Å], forming a polymeric chain. The molybdenum atoms are quasi-octahedrally coordinated by two cis-oxo groups and two bidentate malate ligands via its alkoxy and α-carboxyl groups, while the β-carboxylic and carboxylate groups remain uncomplexed, as the coordination of vicinal carboxylate and alkoxide of homocitrate in FeMo cofactor of nitrogenase. The absolute configuration of the metal center in this S-malato complex is assigned as Λ and the homochirality within the chain is established as a homochiral form ···Λ_S–Λ_S–Λ_S–Λ_S···. It is proposed that the chiral configuration of the metal center in wild-type FeMo-co biosynthesis might be induced by the early coordination of the chiral R-homocitric acid, while a mixture of raceme might be obtained in the biosynthesis of NifV⁻ FeMo-cofactor. The absolute configuration of wild-type FeMo-cofactor is assigned as Δ_R.     

Dynamics of Recognition between tRNA and elongation factor Tu

Elongation factor Tu (EF-Tu) binds to all standard aminoacyl transfer RNAs (aa-tRNAs) and transports them to the ribosome while protecting the ester linkage between the tRNA and its cognate amino acid. We use molecular dynamics simulations to investigate the dynamics of the EF-Tu·guanosine 5′-triphosphate·aa-tRNA^Cys complex and the roles played by Mg²⁺ ions and modified nucleosides on the free energy of protein·RNA binding. Individual modified nucleosides have pronounced effects on the structural dynamics of tRNA and the EF-Tu·Cys-tRNA^Cys interface. Combined energetic and evolutionary analyses identify the coevolution of residues in EF-Tu and aa-tRNAs at the binding interface. Highly conserved EF-Tu residues are responsible for both attracting aa-tRNAs as well as providing nearby nonbonded repulsive energies that help fine-tune molecular attraction at the binding interface. In addition to the 3′ CCA end, highly conserved tRNA nucleotides G1, G52, G53, and U54 contribute significantly to EF-Tu binding energies. Modification of U54 to thymine affects the structure of the tRNA common loop resulting in a change in binding interface contacts. In addition, other nucleotides, conserved within certain tRNA specificities, may be responsible for tuning aa-tRNA binding to EF-Tu. The trend in EF-Tu·Cys-tRNA^Cys binding energies observed as the result of mutating the tRNA agrees with experimental observation. We also predict variations in binding free energies upon misacylation of tRNA^Cys with d-cysteine or O-phosphoserine and upon changing the protonation state of l-cysteine. Principal components analysis in each case reveals changes in the communication network across the protein·tRNA interface and is the basis for the entropy calculations.     

Ligand-induced structural changes in amylose partially complexed with iodine

The influence of complexing agents such as methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol and 2-octanol on the formation of a blue coloured amylose · iodine complex (pH 4.8), under suboptomum concentrations of iodine and in the absence of potassium iodide, is studied by recording the absorbance at 640 nm. A drop in absorbance at 640 nm accompanied by a blue shift in the spectrum (580–640 nm) was observed at higher concentration of the complexing agents. This behaviour of amylose partially complexed with iodine appears to be due to ligand-induced structural changes in the amylose chain. The fall in absorbance at 640 nm observed when the temeprature of amylose · oidine complex in the presence of complexing agents is raised, and the subsequent regeneration of the absorbance on cooling, indicates the possible helix to random coil transition of the amylose chain in an aqueous system.     

Characterisation of the extractable pectins and hemicelluloses of the cell wall of glasswort, Salicornia ramosissima

Cell walls of glasswort (Salicornia ramosissima Woods), a halophytic Chenopodiaceae, prepared as alcohol-insoluble solids, were found to be rich in arabinose, galacturonic acid, glucose and proteins, and contained 0·7% ferulic acid and 3·8% acetic acid. Pectic and hemicellulosic polysaccharides were extracted by cyclohexanediaminotetraacetic acid, hot dilute acid, cold dilute alkali and concentrated alkali (twice), with yields of 2·9, 19·1, 4·7, 7·4 and 1·9% of the alcohol-insoluble solids, respectively. Protein-rich material precipitated upon dialysis. The dialysed fractions were fractionated by ion-exchange chromatography, and the main fractions were analysed by gel-filtration and glycosyl linkage analysis. The hot acid extract contained 46·2% arabinose and 28·9% galacturonic acid, with high degrees of methylation and acetylation (65 and 45, respectively). It could be fractionated into a low-molecular-weight arabinan rich in ferulic acid, and a pectic fraction still relatively rich in neutral sugars. The concentrated alkali extracts were rich in xylose (33·4 and 23·6%, respectively). They were separated by ion-exchange chromatography into a fucogalactoxyloglucan and a glucuronoarabinoxylan.     

Placental vascular responses to leukotriene receptor antagonist FPL 55712

We have previously shown that FPL 55712, a specific leukotriene receptor antagonist, potentiates estrogen induced uterine hyperemia in nonpregnant rabbits. We therefore chose to investigate the vascular responses of pregnant rabbits to leukotriene blockade. Nine unanesthetized animals carrying 46 viable fetuses were used in this study. Regional blood flows were measured by the radioactive microsphere technique. In 5 rabbits control blood flows were measured after vehicle administration and FPL 55712, 1 mg/kg bolus, followed by infusion of 100 ug/kg/min was given via the jugular vein. Regional blood flows were measured again after 10 minutes of infusion. The procedural order was reversed in the remaining 4 animals. Resistance was calculated as mean arterial pressure divided by total flow to an organ. FPL 55712 decreased the blood pressure from 83 ± 2 to 76 ± 3 mmHg (P<.001). Uterine resistance was not significantly changed (387 ± 44 to 362 ± 42 mmHg·ml⁻¹·min·gm), but renal resistance fell from 18.5 ± 1.1 to 15.1 ± 1.2 mmHg·ml⁻¹·min·gm (P<.01). FPL 55712 induced maternal placental vasodilatation with resistance decreasing from 291 ± 33 to 261 ± 31 mmHg·ml⁻¹·min·gm (P<.04). Vehicle administration did not cause dilation in any vascular bed. FPL 55712 appears to be a placental vasodilator whose action is most likely due to receptor blockade of the vasoconstrictive endogenous leukotrienes.     

Rainbow trout (Oncorhynchus mykiss) recombinant IL-1β and derived peptides induce migration of head-kidney leucocytes in vitro

The present work provides the first information concerning the chemoattractant activity of trout recombinant IL-1β and its derived peptides, referred to as P1, P2 and P3. The predicted rainbow trout mature interleukin-1β peptide was produced as a recombinant protein in Escherichia coli. The first peptide, P1, corresponded to fragment 146–157 (YVTPVPIETEAR) of the trout sequence and had an MW of 1·37 kDa. It was equivalent to a region known to be part of the receptor binding domain from the mammalian crystal structure of IL-1β complexed to its receptor. P2 was used as control peptide, consisting of the same 12 amino acids as P1, but arranged in a random sequence (VVEEYIRAPPTT). P3 was synthesised to complex with an adjacent region of the IL-1 receptor, and corresponded to fragment 207–216 (YRRNTGVDIS) of the trout sequence, with an MW of 1·18 kDa. Migration was stimulated when leucocytes were exposed to concentrations of ≥10 ng ml⁻¹rIL-1β. Peptide P3 also induced leucocyte migration, with an optimal dose of 0·25 mm being recorded. While P1 had no effect on cell migration when used alone, synergism was evident as a consequence of combining P1 with a suboptimal dose (0·01 mm) of P3. No synergism occurred when cells were exposed to a combination of P3 and the control peptide P2.     

Optimum conditions for the activation of the alternative complement pathway of a cyprinid fish (Tinca tinca L.). Seasonal variations in the titres

The present work describes the existence of a haemolytic activity in the serum of tench, Tinca tinca, against rabbit red blood cells (RRBC) which was identified as belonging to the alternative complement pathway from the following findings: haemolytic activity disappeared when the serum was heated to 45°C for 20 min; 10 mM EDTA, which chelates Ca²⁺ and Mg²⁺, induced a complete loss of haemolysis; Mg²⁺, but not Ca²⁺, was required for the activity, and the use of sheep red blood cells (SRBC), which have a high content of sialic acid, resulted in the serum activity falling to a very low degree of haemolysis. The ACH50 value (units ml^-1 serum) was defined as the reciprocal of the serum dilution necessary to lyse 50% of 4 × 10⁷ RRBC in a buffered medium of normal ionic strength (μ=0·15) containing 10 mM EGTA and optimum concentrations of Mg²⁺. The optimum conditions for the ACH50 assay were: pH 7·2-7-7; reaction temperature, 15°C; concentration of Mg²⁺, 5 mM; and reaction time, 90 min. Under these conditions, the values of ACH50 in spring, summer, autumn and winter for male tench were 69±13, 91±22, 90±36 and 137±41, and for female tench 100±11, 108±13, 82±12 and 145±17. The highest serum activity was found in the winter, suggesting the importance of this pathway during cold periods when the specific immune response is depressed in ectothermic vertebrates.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Single crystals of dextran: High temperature polymorph

Lamellar single crystals of a high temperature polymorph of synthetic dextran were prepared at temperatures ranging from 120 to 200°C in a mixture of water and polyethylene glycol. Individual crystals with lath-like shapes gave well resolved electron diffraction diagrams from which the reciprocal unit cell parameters a^*, b^* and γ^* could be measured. The direct cell parameters were then determined from a series of electron diffraction diagrams obtained by sequential tilting of the crystal about the b^* axis. This gave a = 0·922 ± 0·001 nm, b = 0·922 ± 0·001 nm, c (chain axis) = 0·78 ± 0·01 nm, α = γ = 90° and β = 91·3° ± 0·5°. The crystal symmetry was P2₁ with b as the unique monoclinic axis. These data coupled with the observed density of the crystals, indicated that the unit cell contained two antiparallel dextran chains of two residues each. When the crystals were grown at temperatures between 90 and 120°C, a percentage of crystals containing both low and high temperature polymorphs were obtained. These mixed crystals had most likely grown in syntaxy.     

Stochastic models of molecular evolution and the estimation of phylogeny and rates of nucleotide substitution in the hominoid primates

We have estimated phylogenetic patterns and rates of nucleotide substitution in the hominoid primates using two different probabilistic models of molecular evolution as applied to three different data sets of nucleic acid sequences. The orang-utan was found to be the out-group of the other hominoids examined. Within the African apes and human clade the sister-group relationship of chimpanzee and human was found to be statistically the best, although the magnitude of the error estimates (a reflection of random statistical fluctuations) makes this conclusion tentative. The ψν-globin data sets were found to be statistically the most consistent and gave estimates of the times of divergence of chimpanzee and human from gorilla and of chimpanzee from human as 7·7 ± 1·5 Ma (Millions of years ago) and 7·4 ± 1·5 Ma respectively, although the speculative nature of these estimates is emphasized. In all cases the calibration point was the assumed divergence of the orang-utan from the remaining hominoids at 14·5 Ma. There was no statistically significant evidence of a slowdown in nucleotide substitution rate for the human lineage, or among the hominoids as a whole with respect to the Old and New World monkeys. We advocate the continued use and development of stochastic models of molecular evolution as a basis for phylogenetic estimation. On this basis one can choose between competing hypotheses of relationship in a statistical manner and can provide estimates of the errors involved in such estimations. The assumptions of all stochastic models are open to test and future refinement.     

The Determination of Phosphorus in Haemophilus influenzae Type b Conjugate Vaccines by Inductively Coupled Plasma-Atomic Emission Spectrometry

This study describes a method for the determination of phosphorus in lyophilized Haemophilus influenzae type b conjugate vaccines by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The concentration of polysaccharide is directly related to the concentration of phosphorus as measured in the laboratory. Phosphorus is present in the polyribosyl-ribitol phosphate (PRP) group of the Haemophilus influenzae type b conjugate vaccine. The repeating unit of PRP is 3-B-D ribose[1-1]ribitol-5-phosphate. Phosphorus in the final container is measured in μ g per dose. The amount of PRP is calculated from this and reported in μ g per dose. The Haemophilus influenzae type b conjugate vaccine was analyzed for phosphorus content within the range of 1·34 to 2·02 μg phosphorus per ml. The relative difference of phosphorus concentrations determined by the ICP-AES method from the phosphorus concentrations determined by the traditional colorimetric molybdate method ranged from 2·2 to 10·6%. Phosphorus spike recovery for the vaccine ranged from 93 to 99% (1·93±0·13 μ g P/ml). The phosphorus determination of NIST SRM 3139 phosphorus spectrometric solution differed by 3·0% from the certified phosphorus value (10·00 mg P/ml).     

The inducible and cytochrome P450-containing dehydroepiandrosterone 7α-hydroxylating enzyme system of Fusarium moniliforme

7α-Hydroxylation of DHEA by Fusarium moniliforme was investigated with regard to inducibility and characterization of the responsible enzyme system. Using GC/MS, the 7-hydroxylated metabolites of DHEA produced after biotransformation by Fusarium moniliforme mycelia were identified. The strain of Fusarium moniliforme hydroxylated DHEA predominantly at the 7α-position, with minor hydroxylation occurring at the 7β-position. Constitutive 7α-hydroxylation activity was low, but DHEA induced the enzyme complex responsible for 7α-hydroxylation via an increase in protein synthesis. DHEA 7α-hydroxylase was found to be mainly microsomal, and the best production yields of 7α-hydroxy-DHEA (28.5 ± 3.51 pmol/min/mg protein) were obtained with microsomes prepared from 18-h-induced mycelia. Kinetic parameters (K_M=1.18 ± 0.035 μM and V_max=909 ± 27 pmol/min/mg protein) were determined. Carbon monoxide inhibited 7α-hydroxylation of DHEA by microsomes of Fusarium moniliforme. Also, exposure of mycelia to DHEA increased microsomal P450 content. These results demonstrated that: (i) DHEA is 7α-hydroxylated by microsomes of Fusarium moniliforme; (ii) DHEA induces Fusarium moniliforme 7α-hydroxylase; (iii) this enzyme complex contains a cytochrome P450.     

Osmotic permeabilities across corneal endothelium and antidiuretic hormone-stimulated toad urinary bladder structures

Osmotic permeabilities of several epithelial structures have been determined with novel optical procedures based on specular microscopy. The osmotic permeabilities of several tissue layers were determined by continuously monitoring the position of the apical tissue borders while an osmotic flow was imposed across those layers. The values found were (in μm/s; mean ± SE): corneal epithelium, 137 ± 30 (n = 5); antidiuretic hormone stimulated toad bladder, 429 ± 64 (n = 6); and corneal endothelium, 711 ± 34 (n = 7). In addition, the osmotically-induced transient change in thickness of the corneal endothelial cells was determined with the help of a computer, and the apparent osmotic permeability measured for the apical membrane was 1420 ± 160 μm/s (n = 5). It is concluded that the osmotic permeability across the endothelial layer is sizably larger than had been previously detected and that osmotic flows across such layer largely traverse the cellular membranes. With osmotic permeability values (per unit of cell membrane area) as large as presently reported, isotonic fluid transport by epithelia can be explained simply on the basis of local osmotic gradients.     

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol have been determined by single-crystal X-ray diffraction studies. The space group of the α-cyclodextrin–p-chlorophenol complex is P2₁2₁2₁ with unit cell dimensions of a=15.299(3), b=24.795(5), c=13.447(5) Å, and that of the α-cyclodextrin–p-cresol complex is P2₁ with unit cell dimensions of a=7.927(7), b=13.568(7), c=24.54(1) Å, β=90.41(8)°. In spite of the similar structures of guest molecules, both complexes have different inclusion modes and packing structures.     

Functional characteristics and responses to adrenergic stimulation of isolated heart preparations from hypothermic and hibernating subjects

Contemporary studies of isolated hearts from hibernators and nonhibernators are presented. Original experiments with isolated perfused hamster hearts are reported. Such hearts can maintain left ventricular function at temperatures as low as 7 °C. Generated left ventricular pressure was 40 ± 9 mm Hg and heart rate was 7 ± 1 beats/min. During cooling heart rate dropped dramatically, coronary flow increased, and ventricular pressure decreased initially, plateaued, and then fell as 7 °C was approached. Norepinephrine can cause increased heart rate and left ventricular pressure at 22 and 7 °C. This positive inotropic and chronotropic response was associated with increased cAMP at 30 sec after stimulation at 22 °C but not at 7 °C. Furthermore. cAMP was also not changed at peak response at 7 °C. Isoproterenol increased cAMP content in 37 °C ventricular slices but not at hypothermic temperatures. Possible mechanisms of nonadenylate cyclase mediation of inotropic and chronotropic responses at 7 °C are discussed.