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Sprouty and Spred {Sprouty-related EVH1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] domain} proteins have been identified as antagonists of growth factor signalling pathways. We show here that Spred-1 and Spred-2 appear to have distinct mechanisms whereby they induce their effects, as the Sprouty domain of Spred-1 is not required to block MAPK (mitogen-activated protein kinase) activation, while that of Spred-2 is required. Similarly, deletion of the C-terminal Sprouty domain of Spred-1 does not affect cell-cycle progression of G0-synchronized cells through to S-phase following growth factor stimulation, while the Sprouty domain is required for Spred-2 function. We also demonstrate that the inhibitory function of Spred proteins is restricted to the Ras/MAPK pathway, that tyrosine phosphorylation is not required for this function, and that the Sprouty domain mediates heterodimer formation of Spred proteins. Growth-factor-mediated activation of the small GTPases, Ras and Rap1, was able to be regulated by Spred-1 and Spred-2, without affecting receptor activation. Taken together, these results highlight the potential for different functional roles of the Sprouty domain within the Spred family of proteins, suggesting that Spred proteins may use different mechanisms to induce inhibition of the MAPK pathway.

The human mitochondrial genome (mtDNA) is a small, circular DNA duplex found in multi-copy in the mitochondrial matrix. It is almost fully transcribed from both strands to produce large polycistronic RNA units that are processed and matured. The 13 mtDNA-encoded polypeptides are translated from mt-mRNAs that have been matured by polyadenylation of their free 3´-termini. A patient with clinical features consistent with an mtDNA disorder was recently shown to carry a microdeletion, resulting in the loss of the termination codon for MTATP6 and in its juxtaposition with MTCO3. Cell lines from this patient exhibited low steady-state levels of RNA14, the bi-cistronic transcript encoding subunits 6 and 8 of the FoF1-ATP synthase, complex V, consistent with a decreased stability. Recent reports of ‘non-stop’ mRNA decay systems in the cytosol have failed to determine the fate of gene products derived from transcripts lacking termination codons, although enhanced decay clearly required the ‘non-stop’ transcripts to be translated. We wished to determine whether functional translation products could still be expressed from non-stop transcripts in the human mitochondrion. Although a minor defect in complex V assembly was noted in the patient-derived cell lines, the steady-state level of ATPase 6 was similar to controls, consistent with the pattern of de novo mitochondrial protein synthesis. Moreover, no significant difference in ATP synthase activity could be detected. We conclude that, in the absence of a functional termination codon, although mitochondrial transcripts are more rapidly degraded, they are also translated to generate stable polypeptides that are successfully integrated into functional enzyme complexes.

Ca2+ signalling in exocrine acinar cells has been shown to be both polarized and pulsatile in all cell types tested, except acutely isolated mouse lacrimal acinar cells. Lacrimal cells are also unusual in that they display a very low sensitivity to Ins(1,4,5)P3 (InsP3) that may be enhanced by placing the cells in primary culture for 12—72h or by intracellular infusion of a low concentration of Ins(1,3,4,5)P4 (InsP4). We have proposed previously that this atypical behaviour stemmed from vesiculation of the endoplasmic reticulum (ER) incurred during isolation of the cells and, furthermore, that time in culture or InsP4 increased sensitivity to InsP3 by increasing ER integrity [Smith, Harmer, Letcher and Irvine (2000) Biochem. J. 347, 77—82]. We have measured the half time for fluorescence recovery after photobleaching (FRAP) of a fluorescent marker (Mag-fluo 4) loaded into the ER lumen in order to determine directly the functional integrity of the ER in lacrimal cells. The half-time for FRAP was increased (indicating a reduction in the functional integrity of the ER) following exposure to anti-microtubule agents (taxol and nocodazole) known to perturb ER structure and decreased (indicating an increase in the functional integrity of the ER) by time in culture and exposure to InsP4. The action of InsP4 was particularly pronounced because it occurred under patch-clamp whole-cell conditions that were themselves found to reduce ER functional integrity. These data show that ER remodelling could be a physiological regulator of Ca2+ signalling and indicate a role for InsP4 in control of this process.

cADP-ribose (cADPr) and nicotinic acid–adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca2+ mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P3 in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca2+-dependent K+ and Cl- currents. Both Ins(1,4,5)P3 and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10–20nM ACh) or second messenger [1–10µM Ins(1,4,5)P3 or cADPr] elicited a sporadic transient activation of the Ca2+-dependent currents; mid-range concentrations [50–500nM ACh, 50µM Ins(1,4,5)P3 or 50–100µM cADPr] elicited high-frequency (approx. 2Hz) trains of current spikes; and high concentrations [more than 500nM ACh, more than 50µM Ins(1,4,5)P3 or more than 100µM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P3R antagonist (heparin). NAADP (50nM to 100µM) did not itself activate the Ca2+-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P3R and RyR are involved in the propagation of the Ca2+ signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.

Earlier reports have shown a remarkable synergism between InsP4 and InsP3 [either Ins(1,4,5)P3 or Ins(2,4,5)P3] in activating Ca2+-dependent K+ and Cl- currents in mouse lacrimal cells [Changya, Gallacher, Irvine, Potter and Petersen (1989) J. Membr. Biol. 109, 85-93; Smith (1992) Biochem. J. 283, 27-30]. However, Bird, Rossier, Hughes, Shears, Armstrong and Putney [(1991) Nature (London) 352, 162-165] reported that they could see no such synergism in the same cell type. A major experimental difference between the two laboratories lies in whether or not the cells were maintained in primary culture before use. Here we have compared directly the responses to inositol polyphosphates in freshly isolated cells versus cells cultured for 6-72 h. In the cultured cells, Ins(2,4,5)P3 at 100 μM produced a robust stimulation of K+ and Cl- currents, as much as an order of magnitude greater than that observed in the freshly isolated cells. However, the freshly isolated cells could be restored to a sensitivity similar to cultured cells by the addition of InsP4 at a concentration two orders of magnitude lower than that of Ins(2,4,5)P3. We discuss the implications of this with respect to the actions of InsP4, including the possibility that disruption of the cellular structure during the isolation of the cells exposes an extreme manifestation of a possible physiological role for InsP4 in controlling calcium-store integrity.

This paper examines the properties in soluble form of two isoforms of starch synthase. One of these, granule-bound starch synthase I (GBSSI), is responsible for the synthesis of amylose inside the amylopectin matrix of the starch granule in vivo. The other, starch synthase II (SSII), is involved in amylopectin synthesis. Both isoforms can use amylopectin and malto-oligosaccharide as substrates in vitro. As well as acting as a substrate for GBSSI, amylopectin acts as an effector of this isoform, increasing the rate at which it elongates malto-oligosaccharides and promoting a processive rather than distributive mode of elongation of these compounds. The affinity of GBSSI for amylopectin as an effector is greater than its affinity for amylopectin as a substrate. The rate and mode of elongation of malto-oligosaccharides by SSII are not influenced by amylopectin. These results suggest that specific interaction with amylopectin in the matrix of the starch granule is a unique property of GBSSI and is critical in determining the nature of its products.

Isoforms of starch synthase belonging to the granule-bound starch synthase I (GBSSI) class synthesize the amylose component of starch in plants. Other granule-bound isoforms of starch synthase, such as starch synthase II (SSII), are unable to synthesize amylose. The kinetic properties of GBSSI and SSII that are responsible for these functional differences have been investigated using starch granules from embryos of wild-type peas and rug5 and lam mutant peas, which contain, respectively, both GBSSI and SSII, GBSSI but not SSII and SSII but not GBSSI. We show that GBSSI in isolated granules elongates malto-oligosaccharides processively, adding more than one glucose molecule for each enzyme-glucan encounter. Granule-bound SSII can elongate malto-oligosaccharides, but has a lower affinity for these than GBSSI and does not elongate processively. As a result of these properties GBSSI synthesizes longer malto-oligosaccharides than SSII. The significance of these results with respect to the roles of GBSSI and SSII in vivo is discussed.

Rat-2 fibroblasts demonstrate specific binding of 125I-labelled rat adrenomedullin (KD = 0.43nM; Bmax = 50fmol/mg of protein) in the absence of 125I-labelled calcitonin-gene-related peptide (CGRP) binding. Therefore Rat-2 cells were used to examine the pharmacology and signal transduction pathways of adrenomedullin receptors. We examined the effects of adrenomedullin, the CGRP receptor antagonist CGRP-(8–37) and the amylin antagonists AC187 and AC253 on receptor binding and cAMP production. AC253, AC187 and CGRP-(8–37) inhibited 125I-adrenomedullin binding, with respective IC50 values of 25±8, 129±39 and 214±56nM. Adrenomedullin dose-dependently increased intracellular cAMP (approximate EC50 = 1.0nM). CGRP-(8–37), AC253 and AC187 antagonized adrenomedullin-stimulated cAMP production at micromolar concentrations. Using kinase-substrate assays, Mono Q FPLC and ‘phospho-specific ’ Western blotting, we found that adrenomedullin alone abolished basal mitogen-activated protein kinase (MAPK) activity and dose-dependently inhibited platelet-derived-growth-factor-stimulated MAPK activity. Radioimmunoassay for adrenomedullin of media from Rat-2 cells showed a linear release of adrenomedullin-like immunoreactivity of 3.1fmol/h per 2×106 cells. Gel-filtration chromatography showed that this adrenomedullin-like immunoreactivity co-eluted with synthetic rat adrenomedullin. Northern blotting with a rat adrenomedullin cDNA probe was used to confirm the presence of adrenomedullin mRNA. However, neither Northern blotting nor reverse transcriptase–PCR showed the presence of the cloned adrenomedullin receptor (L1). We conclude that the Rat-2 cell line expresses a specific adrenomedullin receptor (coupled to cAMP production and regulation of MAPK) and secretes adrenomedullin, which may participate in a regulatory control loop.

Two systems in vitro are described that show insulin-stimulated phosphorylation of the insulin receptor on serine residues. In the first system, insulin receptor was purified partially from Fao rat hepatoma cells by direct solubilization of the cells in Triton X-100 and chromatography on wheat-germ-agglutinin-agarose. Phosphorylation of these preparations with [gamma-32P]ATP in the presence or absence of insulin resulted in 32P incorporation exclusively into phosphotyrosine residues. Serine kinase activity towards the insulin receptor was reconstituted by adding extracts of Fao cells. Prior exposure of the cells to insulin stimulated serine kinase activity towards the insulin receptor in extracts 7.2-fold. A receptor serine kinase activity enhanced by treatment of cells with cyclic AMP analogues was also retained in the reconstituted system. In the second system, insulin receptor and insulin-sensitive serine kinase activity towards the insulin receptor were co-purified from human placenta. The protocol involved preparation of membranes, before solubilization and chromatography on wheat-germ-agglutinin-agarose, by using gentle procedures designed not to disrupt a potentially labile association between the insulin receptor and the serine kinase. Serine kinase activity in these preparations towards the insulin receptor was stimulated up to 10-fold by insulin, and the stoicheiometry of serine phosphorylation was estimated to be approx 0.8 mol/mol of insulin receptor for phosphorylations performed in the presence of insulin. Thus a preparation of insulin receptor is described for the first time that is phosphorylated to high stoicheiometry on serine in an insulin-dependent manner. Conditions that facilitate recovery and assay of serine kinase activity are defined and discussed. These systems provide a basis for characterizing the nature of the insulin-sensitive serine kinase that phosphorylates the insulin receptor, and defining its role in insulin action and control of receptor function.

A modified anterogradely perfused rat heart preparation is described in which all the cardiac output passes through the coronary circulation. Such a preparation develops hypertensive aortic pressures. Hypertensive aortic pressures or insulin stimulate the rate of cardiac protein synthesis and inhibit the rate of protein degradation. Aortic pressure and insulin may be important in the regulation of cardiac nitrogen balance in vivo. By abolishing cardiac prostaglandin synthesis with 4-biphenylacetate, we were able to investigate the possible involvement of prostaglandins in the modulation of protein turnover by pressure overload or insulin. There was no evidence of any involvement. However, insulin stimulated and cycloheximide inhibited cardiac prostaglandin synthesis. These findings are consonant with an enzyme involved in prostaglandin synthesis being short-lived and prostaglandin synthesis being rapidly influenced by activators and inhibitors of protein synthesis and degradation.

An isotope-dilution method is described for the measurement of N tau-methylhistidine release from the perfused rat heart. We argue that release of N tau-methylhistidine is indicative of cardiac actin degradation. N tau-Methylhistidine release is compared with phenylalanine release in the presence of cycloheximide (phenylalanine release being a measure of degradation of mixed proteins). In hearts perfused with glucose plus acetate, the rate of actin degradation was increased by starvation and was not inhibited by insulin. In contrast, the rate of mixed-protein degradation was decreased by starvation and was inhibited by insulin. The fractional rate of degradation of mixed proteins in hearts from fed or starved rats was greater than that for actin. It is suggested that there are at least two pools of intracellular protein, the degradation rates of which differ in terms of their response to insulin and starvation.

The activity of phosphate-activated glutaminase was increased in the kidney, liver and small intestine of rats made diabetic for 6 days with injection of streptozotocin (75 mg/kg body wt.). Insulin prevented this increase in all three tissues. Treatment with NaHCO3, to correct the acidosis that accompanies diabetes, prevented the increase in renal glutaminase activity, but not that in liver or small intestine. Chemically induced acidosis (NH4Cl solution as drinking water) or alkalosis (NaHCO3 solution as drinking water) increased and decreased, respectively, glutaminase activity in the kidney, but were without significant effect on the activity in liver and small intestine. The increase in glutaminase activity in the small intestine during diabetes was due to an overall increase in the size of this organ, and was only detectable when activity was expressed in terms of whole organ, not mucosal scrapings or isolated enterocytes. Prolonged diabetes (40 days) resulted in an even greater increase in the size and glutaminase activity of the small intestine. Despite this marked increase in capacity for glutamine catabolism, arteriovenous-difference measurements showed a complete suppression of plasma glutamine utilization by the small intestine during diabetes, confirming the report by Brosnan, Man, Hall, Colbourne & Brosnan [(1983) Am. J. Physiol. 235, E261-E265].

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.

We investigated the effect of an increase in the left-atrial filling pressure on the rate of left-atrial protein synthesis in the left-side-perfused working rat heart preparation of Taegtmeyer, Hems & Krebs [(1980) Biochem. J. 186, 701-711]. An increase in filling pressure (preload) at a constant aortic pressure (afterload) increased both the intra-atrial pressure and the atrial stroke volume. The aortic pressure (afterload) was held constant. An increase in filling pressure from 5 to 20 cmH2O at an aortic pressure of 70 cmH2O, or an increase in filling pressure of 7.5 to 20 cmH2O at an aortic pressure of 100 cmH2O, significantly stimulated the rates of left-atrial protein synthesis by 30-40%. The stimulation was observed when the rates of protein synthesis were expressed relative to either protein or RNA content. Since perfusate entering the right atrium from the coronary circulation left that atrium passively, the rate of protein synthesis in this compartment can be used as an internal control. Rates of right-atrial protein synthesis were similar to those in the left atria exposed to the lower filling pressures and were unaffected by the increases in left-atrial filling pressure. We suggest that the acute effects of increased left-atrial filling pressure on protein synthesis in that compartment may be important in the development of left-atrial hypertrophy. This condition is seen in patients who have raised pulmonary venous pressures in, for example, mitral stenosis.

The rates of protein synthesis in perfused rat heart ventricular or atrial muscle were measured by incorporation of [U-14C]phenylalanine in the presence of the remaining plasma amino acids. Atrial protein-synthesis rates were about twice the ventricular rates. Atrial RNA contents were also about twice the ventricular contents. Thus the efficiencies of protein synthesis (protein-synthesis rate/RNA) in the two compartments were similar. There were marked differences in ventricular and atrial RNA contents during the course of rat growth. Atrial RNA content was always greater than ventricular content and declined more slowly during growth, producing a 2-fold change in atrial/ventricular RNA-content ratio between the 88 g and 370 g rat groups.

The effects of insulin on glucose uptake and lactate release in the perfused working rat heart have been investigated in three types of preparation: (i) a control low-workload preparation; (ii) an increased-pressure-workload preparation, simulating conditions of aortic pressure encountered in vivo; (iii) an increased-volume-workload preparation, where pumping work done is approximately the same as (ii) but coronary flow is restricted because of the decreased aortic pressure. Insulin stimulated glucose uptake and lactate release in preparations (i) and (ii), but failed to do so in preparation (iii). It was considered possible that preparation (iii) was hypoxic, thus necessitating a maximal stimulation of glucose uptake. This was confirmed by improving cardiac oxygenation by addition of stroma-free haemoglobin to the perfusate in preparation (iii). Under these conditions in the absence of insulin, glucose uptake and lactate release were decreased compared with perfusions in the absence of haemoglobin. Insulin stimulation of both processes was restored. We conclude that the failure of other workers to observe insulin effects on glucose uptake and lactate release under physiological workloads [preparation (ii)] may be a consequence of intracellular hypoxia in their preparations.

Rat hearts were perfused as working preparations by the method of Taegtmeyer, Hems & Krebs [(1980 Biochem. J. 186, 701-711]. In the presence of glucose, insulin significantly inhibited protein degradation at concentrations as low as 50 mu units/ml. Acetate or lactate, when present either as sole fuel for contraction or in combination with glucose, did not inhibit protein degradation. Insulin inhibition or protein degradation was decreased with either lactate as sole fuel. We suggest that the inhibition of protein degradation occurs over the normal range of plasma concentrations of insulin present in vivo and that the presence of glucose may be at least in part necessary for this effect of insulin.

We studied the effects of glucagon, dibutyryl cyclic AMP and dexamethasone on the rate of [14C]pantothenate conversion to CoA in adult rat liver parenchymal cells in primary culture. The presence of 30nm-glucagon increased the rate by about 1.5-fold relative to control cultures (range 1.4–2.3) and 2.4-fold relative to cultures containing 1–3m-i.u. of insulin/ml. The half-maximal effect was obtained at 3nm-glucagon. Dibutyryl cyclic AMP plus theophylline also enhanced the rate by about 1.5-fold. Dexamethasone acted synergistically with glucagon; glucagon at 0.3nm had no effect when added alone, but resulted in a 1.7-fold enhancement when added in the presence of dexamethasone (maximum effect at 50nm). The 1.4-fold enhancement caused by the addition of saturating glucagon concentrations was increased to a 3-fold overall enhancement by the addition of dexamethasone. However, dexamethasone added alone over the range 5nm to 5μm had no effect on the rate of [14C]pantothenate conversion to CoA. The stimulatory effect of dibutyryl cyclic AMP plus theophylline was also enhanced by the addition of dexamethasone. Changes in intracellular pantothenate concentration or radioactivity could not account for the stimulatory effects of glucagon, dibutyryl cyclic AMP or dexamethasone. Addition of 18μm-cycloheximide, an inhibitor of protein synthesis, decreased the rate of incorporation of [14C]pantothenate into CoA and the enhancement of this rate by glucagon and dibutyryl cyclic AMP plus theophylline in a reversible manner. These results demonstrate an influence of glucagon, dibutyryl cyclic AMP and glucocorticoids on the intracellular mechanism regulating total CoA concentrations in the liver.

We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22±0.04 nM (mean±S.E.M.) and a concentration of binding sites (Bmax) of 0.95±0.19 pmol/mg of protein (mean±S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8–37) competed weakly at this site (IC50 > 10 and 601±298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13±0.01 nM; Bmax = 0.83±0.10 pmol/mg of protein) where both CGRP(8–37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15±0.12 and 8.68±0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site–ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8–37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.

The intracellular Ca2+ concentration was measured in single, acutely isolated, mouse submandibular acinar cells loaded with fura-2 AM. All experiments were performed in the absence of extracellular Ca2+ in order to eliminate Ca2+ influx. The microsomal ATPase inhibitor, thapsigargin, was used to release Ca2+ from intracellular stores and simultaneously prevent re-uptake into the stores. Sequential application of thapsigargin (2 μM) and the Ca2+ ionophore ionomycin (500 nM) indicated that thapsigargin was able to mobilize practically all intracellular Ca2+. Furthermore, in comparison with results obtained following inhibition of the plasma membrane Ca2+-ATPase by La3+ (2 mM), it may be shown that slowly unloading the intracellular Ca2+ stores using thapsigargin does not normally cause a massive, cytotoxic, increase in the cytosolic Ca2+ concentration, because Ca2+ is rapidly extruded from the cell across the plasma membrane. Application of a submaximal dose of acetylcholine (500 nM) during the rising phase of the response to thapsigargin caused a 3–4-fold increase in the amplitude of the rise in the cytosolic Ca2+ concentration without any significant alteration of the time course of the response. As thapsigargin alone is capable of mobilizing all releasable Ca2+, this increase in amplitude is most likely the result of inhibition of the Ca2+ extrusion process by acetylcholine.

We previously demonstrated that internalized insulin enters the cytoplasm before accumulating in nuclei of H35 rat hepatoma cells. This finding raises the possibility that insulin may interact with cytosolic proteins in addition to insulin-degrading enzyme (IDE). In the present study, cytosol from H35 hepatoma cells, rat liver or muscle was incubated with A14- or B26-125I-insulin at 4 degrees C for 5-120 min in the absence or presence of 25 micrograms/ml unlabelled insulin. 125I-insulin was cross-linked to cytosolic proteins by disuccinimidyl suberate and analysed by reducing or non-reducing SDS/PAGE and autoradiography. Our results demonstrate the presence of both tissue-specific and common cytosolic proteins which specifically bind insulin. In muscle cytosol, only two proteins of 27 and 110 kDa were specifically labelled with B26-125I-insulin. Seven major bands, of 27, 45, 55, 60, 76, 82 and 110 kDa, were labelled in rat liver cytosol. Detection of cytosolic insulin-binding proteins in H35-cell cytosol was dependent on cell-culture conditions. Labelling in cytosol from serum-deprived cells was decreased or absent compared with cytosol prepared from serum-fed or serum-deprived cells treated with 100 ng/ml insulin for 1 h before preparation of the cytosol, in which six bands, of 32, 41, 45, 55, 82 and 110 kDa, were specifically labelled with B26-125I-insulin. This result suggests that the concentration or binding activity of some cytosolic insulin-binding proteins is rapidly regulated. Labelling of both rat liver and H35 cytosolic insulin-binding proteins was time-dependent, and decreased or disappeared at 120 min in parallel with the degradation of labelled insulin. Fewer bands were specifically labelled with A14-125I-insulin than with B26-125I-insulin. The number of labelled bands observed under reducing and non-reducing conditions was not different in any of the cytosols. The 110 kDa band in all cytosols was identified as IDE by Western-blot analysis; the other proteins did not react with anti-IDE antibody and remain unidentified. 1,10-Phenanthroline (2 mM) increased IDE labelling, but decreased the labelling of 82 and 27 kDa bands. The marked difference in the number of cytosolic insulin-binding proteins in muscle and either H35 cells or liver suggests both that the labelling is specific and that these proteins serve a function and may be involved in some heretofore unknown mechanism of the signalling pathway by which insulin regulates cell growth or differentiation.

The triacylglycerol lipases present in adult Drosophila melanogaster have been investigated. Different lipase activities are present in various tissues in the fly. In particular, an abundant lipase activity is present in the male accessory gland. An esterase null mutant was used to confirm that the enzyme activity was due to a distinct lipase and not non-specific activity from esterase 6 which is also abundant in accessory glands. The properties of the accessory-gland lipase were investigated, and pH optima and substrate utilization suggest that it has some similarities to vertebrate bile-salt-stimulated lipase. Lipase activity is significantly reduced in males and increased in females shortly after mating. This finding suggests that lipase activity is transferred to the female and may be important in mating and reproduction in Drosophila.

The bacterioferritin (BFR) of Escherichia coli is an iron-storage protein containing 24 identical subunits and between three and 11 protohaem IX groups per molecule. Titration with additional haem gave a maximum loading of 12-14 haems per molecule. The e.p.r. spectra and magnetic c.d. spectra of the protein-bound haem show it to be low-spin Fe(III), and coordinated by two methionine residues as previously reported for BFRs isolated from Pseudomonas aeruginosa and Azotobacter vinelandii [Cheesman, Thomson, Greenwood, Moore and Kadir, Nature (London) (1990) 346, 771-773]. A recent sequence alignment indicated that BFR may be structurally related to ferritin. The molecular model proposed for E. coli BFR has a four-alpha-helix-bundle subunit conformation and a quaternary structure similar to those of mammalian ferritins. In this model there are two types of hydrophobic pocket within which two methionine residues are correctly disposed to bind haem. The e.p.r. spectra also reveal a monomeric non-haem Fe(III) species with spin, S = 5/2. On the basis of sequence comparisons, a ferroxidase centre has recently been proposed to be present in BFR [Andrews, Smith, Yewdall, Guest and Harrison (1991) FEBS Lett. 293, 164-168] and the possibility that this Fe(III) ion may reside at or near the ferroxidase centre is discussed.

1. A study was made of the effects of injected l-methionine on the activity of several enzymes of folate metabolism, and on the transport of methotrexate in liver preparations from vitamin B12-deficient ewes and their pair-fed controls receiving vitamin B12. 2. The activities of dihydrofolate reductase (EC 1.5.1.3) and 5-methyltetrahydrofolate–homocysteine transmethylase were significantly decreased in the liver of vitamin B12-deficient animals, but were unaffected by l-methionine. 3. The concentration of S-adenosyl-l-methionine in the liver of deficient animals was about one-half of that in normal animals, and was restored to normal by either vitamin B12 or l-methionine. 4. Methylenetetrahydrofolate reductase (EC 1.1.1.68) from sheep liver was inhibited by S-adenosyl-l-methionine in vitro, but not by concentrations of S-adenosyl-l-methionine found in the liver of vitamin B12-deficient animals after injection of physiological amounts of l-methionine. 5. Pteroylpolyglutamate synthetase activity was significantly increased in the liver of vitamin B12-deficient animals, and was decreased by intravenous injections of l-methionine. 6. l-Methionine injections increased the initial rate of uptake of methotrexate in liver slices from deficient animals and acted synergistically with vitamin B12 to increase the quantity taken up in 40min. The failure of folate metabolism in vitamin B12 deficiency can be satisfactorily explained if l-methionine similarly affects the membrane transport of naturally occurring folates. 7. Further details of the results have been deposited as Supplementary Publication SUP 50028 (4 pages) at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

1. The effects of injected l-methionine (2g every second day for 28 days) on liver folates and other constituents of liver associated with folate metabolism were studied in vitamin B12-deficient ewes and their pair-fed controls receiving vitamin B12. The dose rate of methionine used was sufficient to restore almost to normal the elevated excretion in the urine of formiminoglutamate in the deficient animals. 2. Liver folates active for Lactobacillus casei, Streptococcus faecalis R and Pediococcus cerevisiae were severely depressed in deficient livers and were partly restored by methionine. Analysis of the folates after ion-exchange chromatography showed that the major effect of methionine was to increase the concentrations of tetrahydrofolates and formyltetrahydrofolates. Methyltetrahydrofolates were also increased, but there was no effect of methionine on the small amounts of incompletely reduced folates present in deficient livers. The folates present were predominantly penta-, hexa- and hepta-glutamates whether or not animals received vitamin B12 or methionine. 3. Concentrations of ATP, NAD+, NADH and NADPH were lower in freeze-clamped liver from vitamin B12-deficient sheep than in liver from pair-fed, vitamin B12-treated sheep. These changes were not affected by methionine which was also without effect on the elevated K+/Na+ ratios found in deficient livers. 4. The livers of vitamin B12-deficient animals contained lower concentrations of choline and higher concentrations of lipid than their pair-fed controls. These effects were reversed by methionine.

Starvation (48 h) decreases fructose 2,6-bisphosphate (Fru-2,6-P2) concentrations and the ratio of free to acylated carnitine in hearts of euthyroid rats. These decreases, which are indicative of increased lipid fuel oxidation, are accompanied by decreased rates of glucose uptake and phosphorylation, assessed by using radioactive 2-deoxyglucose. Cardiac concentrations of acylated carnitines were increased at the expense of free carnitine even in the fed state in response to experimental hyperthyroidism, but neither Fru-2,6-P2 concentrations nor rates of glucose utilization were suppressed. Starvation (48 h) did not further increase the proportion of acylated carnitine in the heart in hyperthyroidism, and suppression of Fru-2,6-P2 concentrations and glucose utilization rates by starvation was attenuated. Although glucose utilization rates were decreased, starvation did not decrease immunoreactive GLUT 4 protein concentrations. Furthermore, although hyperthyroidism was associated with a statistically significant (30-40%) increase in relative abundance of GLUT 4 mRNA, the amount of GLUT 4 protein was not increased by hyperthyroidism in either the fed or the starved state. The results demonstrate a significant effect of hyperthyroidism to enhance cardiac glucose utilization in starvation by a mechanism which does not involve changes in GLUT 4 expression but may be secondary to changes in glucose-lipid interactions at the tissue level.

Starvation (48 h) decreased the concentration of mRNA of the insulin-responsive glucose transporter isoform (GLUT 4) in interscapular brown adipose tissue (IBAT) (56%) and tibialis anterior (10%). Despite dramatic [7-fold (tibialis anterior) and 40-fold (IBAT)] increases in glucose utilization after 2 and 4 h of chow re-feeding, no significant changes in GLUT 4 mRNA concentration were observed in these tissues over this re-feeding period. The results exclude changes in GLUT 4 mRNA concentration in mediating the responses of glucose transport in these tissues to acute re-feeding after prolonged starvation.

The validity of the hypothesis that Factor Xa activates Factor V in heparinized plasma was examined by establishing the temporal relationships between Factor V proteolysis and prothrombin consumption in plasma. Factor V was cleaved into Factor Va heavy chain (approx. 110 kDa) and an intermediate (approx. 230 kDa) 30 s after CaCl2 was added to contact-activated plasma (CAP). The larger fragment was converted into Factor V activation peptide (approx. 150 kDa) and Factor Va light chain (approx. 80 kDa) 15 s later. Heparin (approx. 0.05 microM) delayed Factor V proteolysis in CAP by at least 30 s. On supplementing CAP with 1 nM-Factor Xa or 1 nM-thrombin, Factor V was activated 15 s later. Heparin prolonged by 15 s and 45 s the time required to demonstrate Factor V activation in CAP supplemented with Factor Xa and thrombin respectively. Factor V was activated 20 s after tissue factor and CaCl2 were added to plasma, both in the absence and in the presence of approx. 0.05 microM-heparin. In contrast, hirudin and D-Phe-Pro-Arg-CH2Cl (two thrombin inhibitors more effective than heparin) delayed Factor V activation in this plasma by at least 30 s. The fragments of Factor V obtained in heparinized CAP suggest thrombin escapes inhibition and contributes to Factor V activation in that plasma. Production of Factor Va heavy chain and the 230 kDa Factor V fragment invariably preceded efficient prothrombin activation. These observations suggest that heparin, hirudin and D-Phe-Pro-Arg-CH2Cl delay Factor V activation by inhibiting thrombin. The availability of Factor Xa markedly moderates the ability of heparin to inhibit Factor V activation.

The later stages in the pathway of biosynthesis of phycocyanobilin, the chromophore of phycocyanin, were studied by using radiolabelled intermediates. Three possible pathways from biliverdin IX-alpha to phycocyanobilin were considered. 14C-labelled samples of key intermediates in two of the pathways, 3-vinyl-18-ethyl biliverdin IX-alpha and 3-ethyl-18-vinyl biliverdin IX-alpha, were synthesized chemically and were administered to cultures of Cyanidium caldarium that were actively synthesizing photosynthetic pigments in the light. Neither of these two compounds was apparently incorporated into the phycobiliprotein chromophore, suggesting that two of the three pathways were not operative. By elimination, the results imply that the third possible pathway, which involves phytochromobilin, the chromophore of phytochrome, represents the route for biosynthesis of phycocyanobilin. Unfortunately, since 14C-labelled phytochromobilin is not available, no direct proof of this pathway could be obtained. However, if correct, the present interpretation represents a unified pathway for biosynthesis of all plant bilins, via the intermediacy of phytochromobilin.

The small intestine is the major site of glutamine utilization in the mammalian body. During prolonged (40-day) streptozotocin-diabetes in the rat there is a marked increase in both the size and the phosphate-activated glutaminase activity of the small intestine. Despite this increased capacity, intestinal glutamine utilization ceases in diabetic rats. Mean arterial glutamine concentration fell by more than 50% in diabetic rats, suggesting that substrate availability is responsible for the decrease in intestinal glutamine use. When arterial glutamine concentrations in diabetic rats were elevated by infusion of glutamine solutions, glutamine uptake across the portal-drained viscera was observed. The effect of other respiratory fuels on intestinal glutamine metabolism was examined. Infusions of ketone bodies did not affect glutamine use by the portal-drained viscera of non-diabetic rats. Prolonged diabetes had no effect on the activity of 3-oxoacid CoA-transferase in the small intestine or on the rate of ketone-body utilization in isolated enterocytes. Glutamine (2 mM) utilization was decreased in enterocytes isolated from diabetic rats as compared with those from control animals. However, glutaminase activity in homogenates of enterocytes was unchanged by diabetes. In enterocytes isolated from diabetic rats the addition of ketone bodies or octanoate decreased glutamine use. It is proposed that during prolonged diabetes ketone bodies, and possibly fatty acids, replace glutamine as the major respiratory fuel of the small intestine.

DNA sequences encoding ribulose 1,5-bisphosphate carboxylase small subunit precursor from Pisum sativum L. have been transcribed from plasmids containing the SP6 promoter, and translated in a wheat germ cell-free system. The small subunit precursor polypeptide, its N-terminal leader sequence (transit peptide) and the mature small subunit have each been synthesized independently from three different plasmid constructs. The precursor polypeptide is imported into isolated pea chloroplasts and processed to the mature small subunit by a stromal proteinase. The mature polypeptide is neither imported, nor subject to proteolysis by stromal extracts. The transit peptide alone is very rapidly degraded by a stromal proteinase activity which can be inhibited by EDTA or 1,10-phenanthroline. The use of these gene constructs helps to establish the crucial role of the transit peptide in protein import into the chloroplast.

Compared with glucose, lactate + acetate stimulated ventricular protein synthesis in anterogradely perfused hearts from fed or 72 h-starved rats. Stimulation was greater on a percentage basis in starved rats. Atrial protein synthesis was not detectably stimulated by lactate + acetate. Insulin stimulated protein synthesis in atria and ventricles. The stimulation of protein synthesis by lactate + acetate and insulin was not additive, the percentage stimulation by insulin being less in the ventricles of lactate + acetate-perfused hearts than in glucose-perfused hearts. Perfusion of hearts from 72 h-starved or alloxan-diabetic rats with glucose + lactate + acetate + insulin did not increase protein-synthesis rates or efficiencies (protein synthesis expressed relative to total RNA) to values for fed rats, implying there is a decrease in translational activity in these hearts. In the perfused heart, inhibition of protein synthesis by starvation and its reversal by re-feeding followed a relatively prolonged time course. Synthesis was still decreasing after 3 days of starvation and did not return to normal until after 2 days of re-feeding.

Rates of protein synthesis were measured in vivo in several tissues (heart, skeletal muscles, liver, tibia, skin, brain, kidney, lung) of fed rats exposed to O2/N2 (1:9) for 6 h starting at 08:00-11:00 h. Protein synthesis rates were depressed by 15-35% compared with normoxic controls in all of the tissues studied. The decreases were greatest in the brain and the skin. Although hypoxia inhibited gastric emptying, its effects on protein synthesis could probably not be attributed to its induction of a starved state, because protein-synthesis rates in brain and skin were not decreased by a 15-18 h period of starvation initiated at 23:00 h. Furthermore, we showed that protein synthesis was inhibited by hypoxia in the rat heart perfused in vitro, suggesting a direct effect. The role of hypoxia in perturbing tissue nitrogen balance in various physiological and pathological states is discussed.

Protein synthesis and degradation rates in diaphragms from fed or starved rats were compared in vivo and in vitro. For fed rats, synthesis rates in vivo were approximately twice those in vitro, but for starved rats rates were similar. Degradation rates were less in vivo than in vitro in diaphragms from either fed or starved rats.

In vivo, fractional rates of protein synthesis in atrial muscle of hearts taken from fed rats were 70% greater than in ventricular muscle. After 3 days starvation, atrial protein synthesis is inhibited, but the inhibition is less than in ventricles. A crude subcellular fractionation of the aqueous homogenates by centrifugation at 32000g showed that the supernatant and precipitate proteins were synthesized at the same rate in the ventricles. The fractional rates of protein synthesis and RNA/protein ratios in the right ventricle were 10% greater than in the left ventricle. Protein synthesis in both of these regions was inhibited equally by starvation. In vitro, rates of protein synthesis in atria and ventricles of anterogradely perfused rat hearts were stimulated by saturating insulin concentrations and were inhibited by starvation, but the effects in atria were smaller than in ventricles. Rates of protein synthesis in atria in vitro were 80-95% of rates in vivo. The heart therefore shows considerable regional variation in rates of protein synthesis in vivo and in vitro, and the sensitivity of protein synthesis in the various regions to interventions such as insulin and starvation differs.

1. Protein degradation was studied in the glucose (5 mM)-perfused working rat heart preparation of Taegtmeyer, Hems & Krebs [(1980) Biochem. J. 186. 701-711]. 2. The effects of cardiac workload were investigated in three different preparations: (a) control (low workload), (b) increased pressure workload (simulating conditions of aortic pressure in vivo) and (c) increased volume workload. There was no effect of increased workload on protein degradation in preparation (b) or (c) when compared with preparation (a). Insulin inhibited protein degradation in all three preparations. Significantly greater inhibition by insulin was observed in the increased-pressure-workload preparation (b). 3. Hypoxia was induced by the partial replacement of O2 in the gaseous phase by N2. Hearts maintained their cardiac output when O2 content was decreased from 95% to 55% by volume, but the stability of the preparation was less at 50% O2. Lactate output was significantly increased at O2 contents of 65% or less. The rate of protein degradation was not different from control values (95% O2) in perfusions with 65, 55 or 50% O2. 4. We conclude that acutely increased workload or acute hypoxia does not affect protein degradation in the perfused working rat heart when cardiac output is relatively stable.

N-Methylprotoporphyrin IX strongly inhibits synthesis of phycocyanobilin, but not chlorophyll a, in the dark. In the light, both phycocyanin and chlorophyll a synthesis are inhibited in parallel. These results are consistent with the intermediacy of haem in algal bilin synthesis and suggest a control mechanism for chlorophyll a synthesis, previously unknown.

N-Methylprotoporphyrin dimethyl ester inhibits ferrochelatase in isolated membranes of Rhodopseudomonas sphaeroides at low concentrations (around 10 nm). Full inhibition developed after a short lag phase. The inhibition was non-competitive with porphyrin substrate. Addition of inhibitor to growing cultures of Rps. sphaeroides caused a decrease (near 40%) in cytochrome content and a severe inhibition of ferrochelatase; the excretion of haem into the medium by cell suspensions was also severely inhibited. The addition of N-methylprotoporphyrin dimethyl ester to suspensions of photosynthetically competent Rps. sphaeroides Ga caused excretion of Mg-protoporphyrin monomethyl ester. When added to mutants V3 and O1, magnesium divinylphaeoporphyrin a5 monomethyl ester and 2-devinyl-2-hydroxyethylphaeophorbide a were excreted, with maximum effect at around 3 microM-inhibitor in the medium. The results are interpreted to suggest that the inhibitor decreases concentration of intracellular haem, which normally controls the activity of 5-aminolaevulinate synthetase. Unregulated activity of this enzyme leads to overproduction of protoporphyrin, which is diverted to the bacteriochlorophyll pathway. Further control operates at magnesium protoporphyrin ester conversion in normal cells.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.

Detached cucumber (Cucumis sativus L. var. Beit Alpha) cotyledons incubated in darkness with 5-aminolaevulinic acid and either 16O2 air (control) or 18O2 in N2 accumulated protochlorophyllide. This was converted into methyl phaeoporphyrin alpha 5 and analysed by mass spectrometry. The molecular ion of the methyl phaeoporphyrin alpha 5 derived from the 18O2 incubation was 2 mass units greater than that of the control, establishing that the oxo oxygen atom of the isocyclic ring is derived from atmospheric oxygen.

We have proposed previously that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent amplification reactions, and that prothrombinase is formed in heparinized plasma only after Factor Xa activates Factor VIII and Factor V. These propositions were based on the demonstration that both heparin and Phe-Pro-Arg-CH2Cl completely inhibited 125I-prothrombin activation for up to 60 s when contact-activated plasma (CAP) was replenished with Ca2+. Furthermore, the addition of thrombin to CAP before heparin or Phe-Pro-Arg-CH2Cl completely reversed their inhibitory effects. Additional support for the above hypotheses is provided in this study by demonstrating that, when the activity of thrombin is suppressed by heparin (indirectly) or by Phe-Pro-Arg-CH2Cl (directly), exogenous Factor Xa reverses the ability of these two agents to inhibit prothrombin activation. Prothrombin activation was initiated by adding Factor Xa (1 nM) or thrombin (1 or 10 nM) simultaneously with CaCl2 to CAP. In the absence of heparin or Phe-Pro-Arg-CH2Cl, prothrombin activation was seen 15 s later in either case. Heparin failed to delay, and Phe-Pro-Arg-CH2Cl delayed for 15 s, prothrombin activation in CAP supplemented with Factor Xa. In contrast, heparin and Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation for at least 45 s in CAP supplemented with 1 nM-thrombin. Heparin failed to delay prothrombin activation in CAP supplemented with 10 nM-thrombin, whereas Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation in this plasma for 45 s. These results suggest that in CAP: (1) Factor Xa can effectively activate Factor VIII and Factor V when the proteolytic activity of thrombin is suppressed; (2) heparin-antithrombin III is less able to inhibit Factor Xa than thrombin; (3) suppression of the thrombin-dependent amplification reactions is the primary anticoagulant effect of heparin.

Insulin receptor was co-purified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. By using this ‘in vitro’ system, the mechanism of activation of the serine kinase by insulin was explored. Peptide 1150, histone, poly(Glu-Tyr), eliminating Mn2+ (Mg2+ only), treatment at 37 degrees C (1 h), N-ethylmaleimide, phosphate, beta-glycerol phosphate and anti-phosphotyrosine antibody all inhibited insulin-receptor tyrosine kinase activity and the ability of insulin to stimulate phosphorylation of the insulin receptor on serine. Additionally, direct stimulation of the receptor tyrosine kinase by vanadate increased serine phosphorylation of the insulin receptor. Insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the insulin receptor. The activity of the insulin-sensitive receptor serine kinase was not augmented by cyclic AMP, cyclic GMP, Ca2+, Ca2+ + calmodulin, Ca2+ + phosphatidylserine + diolein or spermine, or inhibited appreciably by heparin. Additionally, the serine kinase phosphorylated casein or phosvitin poorly and was active with Mn2+. This indicates that it is distinct from Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, cyclic AMP- and cyclic GMP-dependent protein kinases, casein kinases I and II and insulin-activated ribosomal S6 kinase. Taken together, these data indicate that a novel species of serine kinase catalyses the insulin-dependent phosphorylation of the insulin receptor and that activation of this receptor serine kinase by insulin requires an active insulin-receptor tyrosine kinase.

The concentrations of CoA in the livers of severely vitamin B12-deficient ewes were about 2·6 times those in pair-fed animals treated with vitamin B12. When the feeding rates of the pair-fed animals were closely similar, the concentrations of methylmalonic acid in deficient livers were about twice those in vitamin B12-sufficient livers. The molar concentrations of CoA present were more than three times those of methylmalonic acid in both deficient and treated animals, and it is concluded that the elevated concentrations of CoA in the deficient livers were not primarily due to accumulation of methylmalonyl-CoA.

1. Interactions in the rates of consumption of acetate, propionate and butyrate in sheep liver mitochondria were examined in the presence and absence of l–malate and α–oxoglutarate. 2. Acetate was not consumed in absence of ancillary substrate but utilization of acetate (7.2nmol/min per mg of protein) occurred in the presence of α–oxoglutarate. This consumption was abolished by propionate or butyrate but the presence of acetate did not affect consumption of propionate or butyrate. 3. Propionate consumption (10.1nmol/min per mg of protein) was unaffected by malate but was stimulated by 63% by butyrate or by 180% by α–oxoglutarate. 4. Butyrate consumption (3.3nmol/min per mg of protein) was stimulated by 117% by malate, by 151% by propionate and by 310% by α–oxoglutarate. 5. In the absence of ancillary substrates the maximum rate of total volatile fatty acid utilization (24.7nmol/min per mg of protein) occurred with a mixture of propionate and butyrate. When both propionate and butyrate were present total consumption was not affected by malate but was stimulated by 24% by α–oxoglutarate. With α–oxoglutarate present, propionate and butyrate each decreased the other's consumption by about 26%, but the total utilization was the greatest observed. 6. The inhibition of acetate consumption by propionate or butyrate is unexplained, but the remaining effects are consistent with an interaction of propionate and butyrate through oxaloacetate together with a general limitation imposed by a need for GTP to rephosphorylate AMP formed during activation of the volatile fatty acids.

1. Utilization of propionate by sheep liver mitochondria was stimulated equally by pyruvate or α-oxoglutarate, with formation predominantly of malate. Pyruvate increased conversion of propionate carbon into citrate, whereas α-oxoglutarate increased formation of phosphoenolpyruvate. The fraction of metabolized propionate converted into phosphoenolpyruvate was about 17% in the presence or absence of α-oxoglutarate and about 7% in the presence of pyruvate. Pyruvate consumption was inhibited by 80% by 5mm-propionate. 2. Compared with rat liver, sheep liver was characterized by very high activities of phosphoenolpyruvate carboxykinase and moderately high activities of aconitase in the mitochondria and by low activities of ‘malic’ enzyme, pyruvate kinase and lactate dehydrogenase in the cytosol. Activities of phosphoenolpyruvate carboxy-kinase were similar in liver cytosol from rats and sheep. Activities of malate dehydrogenase and NADP-linked isocitrate dehydrogenase in sheep liver were about half those in rat liver. 3. The phosphate–dicarboxylate antiport was active in sheep liver mitochondria, but compared with rat liver mitochondria the citrate–malate antiport showed only low activity and mitochondrial aconitase was relatively inaccessible to external citrate. The rate of swelling of mitochondria induced by phosphate in solutions of ammonium malate was inversely related to the concentration of malate. 4. The results are discussed in relation to gluconeogenesis from propionate in sheep liver. It is proposed that propionate is converted into malate by the mitochondria and the malate is converted into phosphoenolpyruvate by enzymes in the cytosol. In this way sufficient NADH would be generated in the cytosol to convert the phosphoenolpyruvate into glucose.

1. Metabolism of folate was studied in six ewes in an advanced state of vitamin B12 deficiency as judged by voluntary food intake and in their pair-fed controls receiving vitamin B12. A group of four animals that were maintained throughout the experiment at pasture was also studied. 2. After 34–40 weeks on the cobalt-deficient diet urinary excretion of formiminoglutamate by four deficient animals was about 3.2mmol/day and this was not significantly decreased by injection of three of them with about 4.5μg of [2-14C]folate/kg body weight per day for 5 days. Three days after the last injection retention of [2-14C]folate by the livers of the deficient animals (5.5% of the dose) was lower than that of their pair-fed controls (26% of the dose) but there was no evidence of net retention of injected folate in the livers of either group. Urinary excretion of 14C indicated that renal clearance of folate may have been impaired in very severe vitamin B12 deficiency. 3. As estimated by microbiological assays total folates in the livers of animals at pasture (12.9μg/g) included about 24% of 5-methyltetrahydrofolate as compared with about 72% of a total of 12.5μg/g in three further ewes fed on a stock diet of wheaten hay-chaff and lucerne-chaff. Liver folates of vitamin B12-deficient animals (0.5μg/g) included about 88% of 5-methyltetrahydrofolate as compared with about 51% of a total of 5.2μg/g in pair-fed animals treated with vitamin B12. 4. Chromatography of liver folates of the pair-fed animals permitted quantitative estimates of the pteroylglutamates present. The results showed that the vitamin B12-deficient livers were more severely depleted of tetrahydrofolates and formyltetrahydrofolates than of methyltetrahydrofolates and that as the deficiency developed they were more severely depleted of the higher polyglutamates than of the monoglutamate within each of these classes. Results from animals injected with [2-14C]folate indicated an impairment of the exchange between pteroylmonoglutamates and pteroylpolyglutamates in the livers of deficient animals. 5. In vitamin B12-deficient animals with food intakes below 200g/day some of the liver folates were not completely reduced and some degradation of pteroylpolyglutamates was detected. The latter condition may have been associated with fatty liver. 6. The results are discussed in relation to current theories of vitamin B12–folate interactions.

1. Methods are described for the extraction, separation by ion-exchange chromatography and estimation by microbiological assay of the folates in sheep liver. 2. Injection of [2-14C]-pteroylglutamate into a sheep fed on a stock diet led to extensive labelling of chromatographically separable liver folates. About 12% of the label in the liver could not be extracted by the method used. 3. Liver folates were examined in five ewes fed on restricted amounts of a diet of wheaten hay-chaff and gluten and injected weekly with vitamin B12. Chromatographic separation was followed by microbiological assay with Lactobacillus casei, Streptococcus faecalis R. and Pediococcus cerevisiae both before and after treatment of fractions with conjugase (γ-glutamylcarboxypeptidase). Evidence was obtained that the folates present were predominantly polyglutamate forms of tetrahydropteroylglutamate, 5-methyltetrahydropteroylglutamate and 5- (and 10-) formyltetrahydropteroylglutamates. Differences in the responses of the assay organisms permitted quantitative distinction between these three main classes of folates. 4. Methyltetrahydrofolates were eluted in seven successive peaks that were separated by constant increments in the logarithm of eluant [Pi]. A similar relationship existed for seven successive peaks of tetrahydrofolate and may also have existed for each of the two series of formyltetrahydrofolates. 5. Based on these and other observations it is proposed that sheep liver folates consist predominantly of the mono- to hepta-glutamates of each of the reduced pteroates identified. The methods employed allowed quantitative determinations to be made of most of the folates present. The predominant forms were hexaglutamates. 6. Four components active for L. casei were detected that could not be identified. Three of them were polyglutamates.

1. Sephadex G-15 was used to separate pteroylmonoglutamates from corresponding polyglutamate derivatives. 2. Pteroylpolyglutamates were formed when 5-formyltetrahydro[2-14C]pteroylglutamic acid, 5-[methyl-14C]tetrahydropteroylglutamic acid or tetrahydro[2-14C]pteroylglutamic acid was incubated at pH8.4 with ATP, MgCl2, KCl, l-glutamic acid and sheep liver cytosol. The γ-glutamyl side chain appeared to be lengthened by the stepwise addition of single glutamate moieties. 3. The subcellular distribution of pteroylpolyglutamates paralleled that of pteroylpolyglutamate synthetase activity, and followed the order cytosol>‘nuclear’ fraction>microsomal fraction>mitochondria.