Search

GPI-PLD [glycosylphosphatidylinositol (GPI)-specific phospholipase D (PLD)] is a secreted mammalian enzyme that specifically cleaves GPI-anchored proteins. In addition, the enzyme has been shown to cleave GPI anchor intermediates in cell lysates. The biosynthesis of the GPI anchor is well characterized; however, the mechanisms by which the levels of GPI anchor intermediates are regulated are still unknown. To investigate whether GPI-PLD plays a role in this regulation, we isolated stable HeLa cells overexpressing the enzyme. GPI-PLD-HeLa (GPI-PLD-transfected HeLa) cells showed a 3-fold increase in intracellular GPI-PLD activity and drastically decreased the levels of GPI-anchored proteins when compared with untransfected HeLa controls. Intracellular cleavage of GPI-anchored proteins has been suggested to occur early in the secretory pathway and, in agreement with this proposal, GPI-PLD activity in GPI-PLD-HeLa cells was detected not only in the endoplasmic reticulum and Golgi apparatus, but also in the plasma membrane. The enzyme was also active in lipid rafts, membrane microdomains in which GPI-anchored proteins and GPI anchor intermediates are concentrated, indicating that intracellular GPI-PLD cleavage may also occur in this compartment. Pulse–chase paradigms revealed the turnover rate of the last intermediate of the GPI anchor pathway in GPI-PLD-HeLa cells to be accelerated compared with the controls. Furthermore, 1,10-phenanthroline, a GPI-PLD inhibitor, reversed this effect. Our studies demonstrated that GPI-PLD can cleave not only GPI-anchored proteins, but also GPI anchor intermediates intracellularly. This observation opens the possibility that GPI-PLD can influence the steady-state levels of GPI-anchored proteins by hydrolysing the anchor before and after its attachment to proteins.

The nucleolus is a plurifunctional, nuclear organelle, which is responsible for ribosome biogenesis and many other functions in eukaryotes, including RNA processing, viral replication and tumour suppression. Our knowledge of the human nucleolar proteome has been expanded dramatically by the two recent MS studies on isolated nucleoli from HeLa cells [Andersen, Lyon, Fox, Leung, Lam, Steen, Mann and Lamond (2002) Curr. Biol. 12, 1–11; Scherl, Coute, Deon, Calle, Kindbeiter, Sanchez, Greco, Hochstrasser and Diaz (2002) Mol. Biol. Cell 13, 4100–4109]. Nearly 400 proteins were identified within the nucleolar proteome so far in humans. Approx. 12% of the identified proteins were previously shown to be nucleolar in human cells and, as expected, nearly all of the known housekeeping proteins required for ribosome biogenesis were identified in these analyses. Surprisingly, approx. 30% represented either novel or uncharacterized proteins. This review focuses on how to apply the derived knowledge of this newly recognized nucleolar proteome, such as their amino acid/peptide composition and their homologies across species, to explore the function and dynamics of the nucleolus, and suggests ways to identify, in silico, possible functions of the novel/uncharacterized proteins and potential interaction networks within the human nucleolus, or between the nucleolus and other nuclear organelles, by drawing resources from the public domain.

The N-terminal sequences of mouse laminin α3B and α5 chains have been completed and demonstrate the presence of a signal peptide followed by a complete laminin N-terminal (LN) module (domain VI). These signal peptides were released after recombinant production of larger fragments comprising domains VI/V (45–65kDa) from this region yielding properly folded proteins, which were secreted from HEK-293—EBNA cells. Pepsin digestion of these fragments yielded products of 25–35kDa, which consisted only of domain V. The αVI/V fragments were able to inhibit self-assembly of laminin-1, with those from the α3B and α5 chains being more active than those from α1 and α2 chains. Domain V fragments, however, showed a reduced activity, indicating the major contribution of the LN module in inhibition. These interactions were confirmed by surface-plasmon-resonance assays demonstrating moderate affinities (Kd = 0.02 to > 6μM) for the binding to laminin-1. This indicated that laminins containing α3B or α5 chains should also be able to form non-covalent networks by polymerization. The LN modules also showed heparin binding in affinity chromatography, which was strongest for α1/α2, moderate for α3B, whereas no binding was observed for α5. They all bound to heparan sulphate chains of perlecan and to sulphatides, with a lower variability in binding activity. Specific antibodies were raised against α3BVI/V and α5VI/V and were shown to stain basement membrane zones in various mouse tissues. These antibodies also allowed the identification of a new laminin assembly form 5B consisting of α3B, β3 and γ2 chains.

The signalling mechanisms involved in the induction of nitric oxide synthase and L-arginine transport were investigated in bacterial lipopolysaccharide (LPS)- and interferon-γ (IFN-γ)-stimulated rat cultured aortic smooth muscle cells (RASMCs). The expression profile of transcripts for cationic amino acid transporters (CATs) and their regulation by LPS and IFN-γ were also examined. Control RASMCs expressed mRNA for CAT-1, CAT-2A and CAT-2B. Levels of all three transcripts were significantly elevated in activated cells. Stimulated CAT mRNA expression and L-arginine transport occurred independently of protein kinase C (PKC), protein tyrosine kinase (PTK) and p44/42 mitogen-activated kinases (MAPKs), but were inhibited by the p38 MAPK inhibitor SB203580, which at 3 μM caused maximum inhibition of both responses. Induction of NO synthesis was independent of p44/42 MAPK activation and only marginally dependent on PKC, but was attenuated markedly by the PTK inhibitors genistein and herbimycin A. SB203580 differentially regulated inducible NO synthase expression and NO production, potentiating both processes at low micromolar concentrations and inhibiting at concentrations of ⩾ 1 μM. In conclusion, our results suggest that RASMCs constitutively express transcripts for CAT-1, CAT-2A and CAT-2B, and that expression of these transcripts is significantly enhanced by LPS and IFN-γ. Moreover, stimulation of L-arginine transport and induction of NO synthesis by LPS and IFN-γ appear to be under critical regulation by the p38 MAPK, since both processes were significantly modified by SB203580 at concentrations so far shown to have no effect on other signalling pathways. Thus, in RASMCs, the p38 MAPK cascade represents an important signalling mechanism, regulating both enhanced L-arginine transport and induced NO synthesis.

Glycosylphosphatidylinositol (GPI)-anchored proteins are resistant to solubilization with Triton X-100 at 4 °C, and they can be recovered in Triton-insoluble membranes (TIMs) that float to a characteristic buoyant density. Because the GPI structure itself has been shown to target GPI-anchored proteins to TIMs, we investigated the association of GPI-anchor intermediates with TIMs. GPI-anchor biosynthesis involves a pathway of some 10 steps that take place in the endoplasmic reticulum (ER). These intermediates include glucosaminyl-acylphosphatidylinositol [GlcN-(acyl)PI] and later mannosylated GPIs, denoted H6, H7 and H8, that are present not only in the ER but also in other cell compartments, including the plasma membrane. At least two-thirds of the GlcN-(acyl)PI in HeLa D cells and mannosylated GPIs in K562 cells were found in TIMs. Although previous reports have considered TIMs to be derived primarily from the plasma membrane, we recovered TIMs from subcellular fractions enriched in ER membranes. The ER marker calnexin and GPI-anchored proteins as well as N-acetylglucosaminyl-phosphatidylinositol and mannosylated GPIs were present in ER-TIMs. Interestingly, GlcN-PI and H7 were less enriched in ER-TIM than the other GPIs, suggesting that ER-TIMs might reflect a compartmentalization of the GPI-anchor biosynthetic pathway in the ER.

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca2+-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca2+/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca2+-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5′-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca2+-dependent and Zn2+-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mitochondrion, is diminished in animal models of diabetes. Studies with purified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydrolipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdomain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inactivation of the complex. By combining molecular modelling, site-directed mutagenesis and biophysical characterizations, we have also identified two amino acid residues in this subdomain (Ile229 and Phe231) that largely determine the magnitude of this effect.

We have applied the paired-tracer dilution method to the study of transport processes in a mixed mammalian muscle preparation, the perfused rat hindlimb. The method is suitable for the characterization of the kinetic parameters of sugar and amino acid transport and its regulation by hormones, contractile activity, hypoxia, etc. Insulin stimulates sugar transport by increasing the Vmax. of the process 2-3 fold, but its affinity is unchanged. Starvation increases the affinity of sugar transport in perfused skeletal muscle.

1. Copper concentrations were low in many organs of Moblo/Y mice, but very high in the gut. Absorption of 64Cu was seen to be very low when related to the absorption of cyano[57Co]cobalamin. The results in Moblo/+ mice were intermediate. 2. Copper therapy temporarily ameliorated many effects of the mutation in Moblo/Y mice, but did not improve the rate of weight gain as has been achieved previously in Mobr/Y mice. Lower capacity for a ‘depot dose’ effect at the site of injection may explain the difference. 3. The distribution of 64Cu after administration into the bloodstream of Moblo/Y mice altered from an initially normal state to one that resembled the abnormal distribution of pre-existing copper by 48 h. This indicated that the later mechanisms of copper distribution were at fault. Moblo/+ mice were equally affected. 4. The alteration of copper homoeostasis in blotchy mice was similar to that observed in brindled mice previously and in the present studies, although generally less severe. This is consistent with allelism of the two mutations.

The active forms of all of the matrix metalloproteinases (MMPs) are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Inhibition represents a major level of control of MMP activity. A detailed knowledge of the mechanisms controlling TIMP gene expression is therefore important. We have isolated a genomic clone of the human TIMP-1 gene. A 3 kbp XbaI fragment has been sequenced; this fragment contains 1718 bp 5′ flanking sequences, exon 1, a 929 bp intron 1 and part of exon 2. Computer analysis reveals 10 consensus sequences for Sp1, six for activating protein 1 (AP-1), six for polyoma enhancer A3 (PEA3), 12 for AP-2 and five CCAAT boxes. The region hybridizing with a murine TIMP-1 promoter fragment has been subcloned and analysed further. RNase protection identifies six transcription start points, making exon 1 up to 48 bp in length. Transient transfection of promoter–chloramphenicol O-acetyltransferase reporter constructs into primary human connective tissue fibroblasts shows that a 904 bp fragment that hybridizes to a murine TIMP-1 promoter fragment contains a functional promoter. Constructs of -738/+95 to -194/+21 are inducible with serum or phorbol ester to a similar extent to the endogenous TIMP-1 gene. These results and further mapping with 5′ deletion mutants from the -738/+95 region have demonstrated that an AP-1 site at -92/-86 is essential for basal expression of the gene. Point mutations within this region have further confirmed the role of this site, along with a more minor role for a neighbouring PEA3 site, in basal expression. Deletions from the 3′ end also implicate a region across the exon 1/intron 1 boundary and especially +21 to +58 in basal expression. The +21/+58 region contains a putative binding site for the transcription factor leader-binding protein 1 (LBP-1). Gel-shift analysis shows that protein binds specifically to this region, but competition studies suggest that it is unlikely to be LBP-1.

Creatine and its phosphorylated form play a central role in the energy metabolism of muscle and nerve tissues. l-Arginine: glycine amidinotransferase (AT) catalyses the committed step in the formation of creatine. The mitochondrial and cytosolic forms of the enzyme are believed to derive from the same gene by alternative splicing. We have expressed recombinant human AT in Escherichia coliwith two different N-termini, resembling the longest two forms of the enzyme that we had isolated recently from porcine kidney mitochondria as a mixture. The enzymes were expressed with N-terminal histidine tags followed by factor Xa-cleavage sites. We established a new method for the removal of N-terminal fusion peptides by means of an immobilized snake venom prothrombin activator. We identified cysteine-407 as the active-site residue of AT by radioactive labelling and isolation of labelled peptides, and by site-directed mutagenesis of the protein.

We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA‘BB’. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (BSP-A1/A2) and BSP-A3. However, unlike the bovine proteins which bind quantitatively to a heparin-Sepharose column, stallion HSP-1 was recovered in both the flow-through and the heparin-bound fractions. Structural analysis showed that the two HSP-1 forms contain identical polypeptide chains which are differently glycosylated. Moreover, size-exclusion chromatography showed that heparin-bound HSP-1 associates with HSP-2, another major seminal plasma protein, into a 90 kDa product, whereas the non-heparin-bound glycoform of HSP-1 is eluted as a monomeric (14 kDa) protein. This suggests that glycosylation may have an indirect effect on the heparin-binding ability of HSP-1 through modulation of its aggregation state. On the other hand, both glycoforms of HSP-1 displayed gelatin-binding activity, indicating that the molecular determinants for binding heparin and gelatin are different.

In its native state, recombinant human-stem-cell-factor (SCF) dimer can spontaneously and rapidly undergo hybridization when two different SCF dimer species are incubated together. SCF species differing in molecular charge, e.g., a wild-type SCF form and a variant with Asp at position 10 instead of Asn, were used in the hybridization studies; the original species and newly formed dimer hybrid can be separated and quantified by cationic-exchange h.p.l.c. The hybridization reaches an equilibrium where the ratio of hybrid dimer to each of the original species is 2. Kinetic studies of the initial rate of hybridization enable a rate constant for monomer dissociation to be determined. This rate constant is influenced by pH, temperature and salt concentration. The pH and salt effects suggest that salt bridges between charged amino acids at the monomer-monomer interface may be present. From the temperature effects, the activation energy for monomer dissociation was determined to be 85.6 kJ/mol, which is typical for oligomeric proteins. Heavily glycosylated recombinant SCF from Chinese-hamster ovary cells exchanged equally well with the bacterially derived non-glycosylated SCF, indicating that the attached carbohydrate moieties had no effect on monomer exchange.

The human integrin glycoprotein (GP)IIb/IIIa plays a central role in haemostasis as an inducible receptor for fibrinogen and other RGD-containing adhesive proteins at the platelet plasma membrane. Expression of the fibrinogen receptor on platelet activation involves conformational changes in the quaternary structure of GPIIb/IIIa. Little is known, however, about the nature of this conformational transition. Given that isolated GPIIb/IIIa contains a mixture of RGD-binding and non-RGD-binding heterodimers, we used limited proteolysis as a tool for investigating the structural differences between the two conformers. Comparison of their fragmentation patterns shows that, whereas in the non-RGD-binding form of GPIIb/IIIa the N-terminal half of the heavy chain of GPIIb (GPIIbH) and the central region of GPIIIa are cleaved by endoproteinase Arg-C, these domains associate tightly with one another in the RGD-binding GPIIb/IIIa and are thus protected from proteolysis. In addition, the C-terminal half of GPIIb becomes more susceptible to degradation in the non-RGD-binding GPIIb/IIIa conformer. Our interpretation, in the context of available structural and functional data, is that a major relative reorientation of the GPIIbH and GPIIIa extracellular domains takes place along the subunit interface during the conformational transition of the platelet integrin.

Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Bacterial lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production, which was further increased in the presence of interferon-gamma. Nitrite synthesis was absolutely dependent on extracellular L-arginine and inhibited in the presence of L-lysine or L-ornithine. In unactivated J774 cells L-arginine transport was saturable, with an apparent Km of 0.14 +/- 0.04 mM and Vmax. of 15 +/- 2 nmol/h per 10(6) cells. LPS (1 microgram/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vmax. increased to 34 +/- 2 nmol/h per 10(6) cells. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of NO.

1. Duodenal injection of 64Cu in treated adult mutant mice (Mobr/y) revealed severe malabsorption of copper. In suckling mutants, malabsorption was less severe, owing to delayed absorption between 2 and 5 h after injection. Pinocytosis at the distal small intestine seems the likely explanation for this difference, and this is supported by results of ileal injection of radioisotope in the suckling mice. 2. The distribution of 64 Cu in various organs was measured in suckling normal, mutant and heterozygote mice and in adult normal and mutant mice during 48 h after intracardiac injection. Excessive accumulation of radioisotope was observed in most extrahepatic organs of mutant and heterozygote mice and was most pronounced in kidney. This could not be explained by initial copper deficiency. The livers of suckling mutant and heterozygote mice lost radioisotope rapidly after normal initial uptake. This pattern was not seen in adult mutants.

Copper therapy was applied to brindled mouse mutants, which suffer from lethal hypocupraemia, by using cuprous and cupric solutions. The method of treatment was a single subcutaneous injection of 50 microgram of copper at 7 days of age. Early effects of the dose were: prevention of the tremors and spasms seen in untreated mutants, raising to normal and near-normal of caeruloplasmin oxidase and lysyl oxidase activities and pigmentation of skin and fur. Growth of mutants was retarded up to 23 days of age, but thereafter they rapidly gained weight to be nearly normal by 60 days of age. At 3 days after injection, copper concentrations in previously deficient mutant organs apart from liver were at least as much as those of treated normals, which had remained unchanged. Copper in mutant livers had increased only slightly in comparison with the normal control. A state of copper deficiency recurred in mutant tissues by 25 days after injection. A solution of Cu+, retained as such by an alkyl polyether, and sebacic acid resulted in greater growth rates after 23 days than did three other copper treatments. Cu+ may have resulted in an improved growth response owing to it being more readily metabolized than C12+. Delayed release of copper from the site of injection may have played an important role.

The copper content of various organs of ‘brindled’ female heterozygotes and male mice affected by this X-linked mutation are documented at the last day of intrauterine development, at 1 day after birth and at 11 days of age. The findings indicate defective placental transfer of copper in utero, and an even more marked defect in intestinal absorption of copper after birth. In addition there is an abnormal distribution of copper among the tissues of the body once it is absorbed. The mutation produces abnormal accumulation of copper in kidney, in gut mucosa and in testis, whereas liver, brain, plasma and most other organs show diminished copper concentrations. The intestinal malabsorption of copper is accompanied by accumulation of abnormal amounts of the metal in the intestinal-mucosa cells. Copper concentrations in both mucosa and luminal contents rise progressively from duodenum to ileum. Defective upper-intestinal absorption, consequent progressive increase in luminal copper concentration and pinocytosis in the ileum would seem to explain the findings. Radioisotopic studies eliminated the possibility of excessive excretion of copper in bile or across the intestinal mucosa. Detailed comparison with findings in humans with Menkes' syndrome is difficult because of the different stages of development at which the studies have been performed, but the results seem in general to conform very satisfactorily. Those differences seen are probably explicable by known species differences. All the findings are in accord with a hypothesis that the basic defect involves accumulation and retention of copper in the cells of affected tissues such as kidney, gut mucosa and placenta.

The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP–PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP–PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4–6 mg of more than 95% pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34% α-helical and 33% β-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP–PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex.

We have demonstrated previously that in bovine adrenal glomerulosa cells, phospholipase D (PLD) activity can indirectly result in the generation of sn-1,2-diacylglycerol (DAG) through its production of phosphatidic acid (PA) and the subsequent action of PA phosphohydrolase. Furthermore, the PLD-generated DAG can trigger aldosterone secretion. Therefore, we characterized PLD activation by two agonists, angiotensin II (Ang II) and carbachol, to determine if the activity of the enzyme might underlie sustained aldosterone secretion. We determined that Ang II-induced PLD activation occurred via the angiotensin-1 receptor (AT1), and that a specific AT1 antagonist, losartan, inhibited this activation, whereas the same concentration of the AT2-specific antagonist, PD 123319, had no effect. Ang II activated PLD with a dose dependence similar to that observed for aldosterone secretion, with slight increases in activity induced by 0.1 nM Ang II and maximal activation at 10 nM. We also found that Ang II induced a sustained activation of PLD, but that the effect of carbachol, a stable analogue of acetylcholine, was transient; PLD activity increased within 5 min of exposure to carbachol but then ceased by 15 min. Higher carbachol concentrations were also unable to sustain PLD activation. These results suggest that the Ang II-elicited activation of PLD is associated with a sustained increase in aldosterone secretion from glomerulosa cells and further provide the first evidence, to our knowledge, of differences in the kinetics of PLD activation in response to two physiologically relevant agonists. Finally, we speculate that this disparity correlates with different functional responses induced by the two agents.

The globular domain IVa (about 250 residues) of the laminin α1 chain was obtained in recombinant form from mammalian cell clones. It was prepared either with (α1IVa-R) or without (α1IVa) an adjacent cell-adhesive RGD site which seems to be masked in laminin-1. The recombinant products could be visualized as globular structures by rotary shadowing, were resistant to trypsin and shared immunological epitopes with laminin-1, indicating folding into a native structure. Sequence analysis of pepsin fragments demonstrated the insertion of the globular domain into an epidermal growth factor-like scaffold which is characteristic of the extracellular laminin domain IV (L4) module. Only little immunological cross-reaction was found, however, with other L4 modules from perlecan and different laminin isoforms. Fragment α1IVa-R, but not fragment α1IVa, bound to αVβ3 integrin, although to a distinctly lower level than a laminin fragment where the RGD site is fully exposed. The fragments also had no or only little cell attachment activity. This confirmed previous predictions that the globular domain α1IVa masks the RGD site in laminin-1. Domain α1IVa showed, in addition, a weak binding activity for the basement-membrane protein fibulin-1.

The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system. Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II). CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates. Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus. The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated. Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively.

Human platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the subunits of the Ca(2+)-dependent heterodimer GPIIb/IIIa, which belongs to the integrin family of phylogenetically related receptors mediating a wide variety of cell-cell and cell-substratum interactions. GPIIb/IIIa plays a central role in haemostasis as a receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. The covalent structure of the subunits is largely known; however, the tertiary and quaternary structures of the heterodimer remain to be determined. To this end, our approach consisted of limited proteolysis of the isolated heterodimer with proteinases of different specificities, followed by protein-chemical and immunochemical analyses of the peptide fragments within each isolated proteolytic product. From the information obtained, we have drawn a rudimentary map which outlines the demarcation of compact domains and the subunit peptide stretches carrying the sequences putatively involved in intrachain, intrasubunit and intersubunit non-covalent connectivity in the heterodimer. Three compact domains have been well defined: one in the heavy (H) chain of GPIIb [GPIIbH-(600-700)], and two in GPIIIa, the N-terminal [GPIIIa-(1-52)] and the core [GPIIIa-(423-622)] domains. Between the latter two domains there is a proteolysis-susceptible region, which is partly involved in ligand binding [GPIIIa-(100-220)] and partly implicated as being in teh subunit interface of the heterodimer. Contrary to GPIIIa, GPIIbH is highly susceptible to proteolysis all along its sequence. Equally susceptible are the extracellular end of the transmembrane segment of both GPIIIa and the light (L) chain of GPIIb (GPIIbL), and the N-terminal end of GPIIbL. Three sequence stretches along the C-terminal half of GPIIbH, one sequence stretch in GPIIbL and three sequence stretches within the GPIIIa-(217-421) region were putatively involved in the subunit interface of the heterodimer. Most likely, the N-terminal end of GPIIbL is folded over the N- and C-terminal regions of GPIIbH, and the N-terminal end of GPIIbH is folded against the GPIIbH-(600-700) domain. This map of GPIIb/IIIa does not fit the current accommodation of the amino acid sequence of GPIIb and GPIIIa in the head/two-tails image of the heterodimer obtained by metal-rotary-shadowing electron microscopy.

The expression of hepatic cytosolic malic enzyme in the developing rat has been studied by molecular-biological techniques. Malic enzyme mRNA was barely detectable throughout the neonatal period, but increased to significant levels immediately before weaning. Northern-blot analysis demonstrated that the two major malic enzyme mRNA species displayed non-co-ordinate control during development, with the 2.0 kb form accumulating to a greater extent than the 3.1 kb form. A novel 1.6 kb mRNA species was found to predominate in foetal samples. Tri-iodothyronine treatment of neonatal rats caused premature induction of all three malic enzyme mRNA species. Dietary studies also showed precocious induction of the mRNA with diets high in carbohydrate, but not with those high in fat.

In contrast with the complex series of splicing choices that generate the various membrane-associated isoforms of the neural cell-adhesion molecule alternative splicing of 5′ exons does not contribute to additional molecular diversity. A single regulatory unit in genomic DNA, mapping to a 5 kb restriction-endonuclease-HindIII fragment, controls the expression of all major RNA size classes. DNA sequence analysis of a 2 kb fragment spanning the two major identified transcriptional initiation sites (194 and 188 bp from the ATG codon) and translation start codon indicates that the regulatory unit does not possess classical TATA or CCAAT motifs. The region of the putative promoter exhibits a GC-rich content and a high frequency of the dinucleotide CpG, both characteristics of a HTF(HpaII tiny fragments)-island. Introduction of deletion-mutant chimaeric-gene constructs into human and rodent N-CAM-expressing cell lines defines an active promoter region of 467 bp (-144 to -611 bp from the ATG codon). This region of genomic DNA contains consensus sites for the interaction of known transcriptional factors.

Individual pools of intracellular inorganic phosphate (Pi) can be observed in the dark in intact cells, protoplasts and chloroplasts from photosynthetic tissue by using 31P nuclear magnetic resonance (n.m.r.). Estimates for the pH of vacuolar and extravacuolar compartments are reported although it is shown that intracellular pH is determined by the pH of the suspending medium. Mannose treatment of asparagus (Asparagus officinalis) cells and spinach (Spinacia oleracea) protoplasts results in the inhibition of photosynthesis. The mechanism of mannose phosphate sequestration of free Pi is supported by the 31P n.m.r. spectra of mannose-treated tissue. There is a fundamental difference in 31 P n.m.r. spectra of mannose-treated spinach protoplasts and asparagus cells, reflecting a difference in the availability of vacuolar Pi for cellular metabolism in these species. The 31P n.m.r. spectrum of intact spinach chloroplasts is reported.

1. The kinetics of calcium transport were studied in unmated (oestrous) and pregnant rabbits in the first half of gestation, with the aim of establishing evidence of hormonal (ovarian) influence on the pattern of transport. 2. The following tissues were examined at short- (45min and 2h) and long-duration (4, 16 and 48h) intervals after parenteral administration of 45Ca or 47Ca: maternal blood plasma, endometrium, uterine fluid, placental tissues, two developmentally disparate stages of rabbit conceptus, namely the unattached blastocyst and the early post-implantation foetus, and bone (femur). 3. Marked variability in calcium content characterized rabbit tissues and body fluids. 4. Compartmental analysis was applied to measurements of specific radioactivity. Oestrous endometrium had the largest rapidly exchanging calcium fraction (turnover time of 12min) and the highest value for calcium flux (500µg of Ca exchanged/h per g fresh wt. of tissue). A marked downward gradient in values of flux existed between the progestational endometrium, uterine fluid and blastocyst; there was a similar gradient between placental tissues and foetus. 5. An hormonal influence on calcium transport was evident in (i) the decrease in specific radioactivity of rabbit blood plasma with advancing pregnancy, (ii) the extraordinarily rapid calcium transport between blood plasma and endometrium, especially in the oestrous stage, and (iii) the effectiveness of ovarian hormone substitution in ovariectomized rabbits. 6. The very low specific radioactivity recorded for bone indicated that only a minute fraction of its calcium was exchanging with that of blood plasma under the experimental conditions examined. 7. The rate of uptake of 45Ca by rabbit blastocysts growing in vitro was one-tenth of that of 22Na, or that recorded for calcium in vivo. 8. Inhibition of carbonic anhydrase activity with acetazolamide in vivo, in maternal erythrocytes, endometrium and placental tissues, produced no appreciable changes in calcium uptake in these tissues or other systems examined as a routine on either day 6 or days 12–14 of gestation.

1. The existing procedures for extraction of oxidized and reduced nicotinamide coenzymes were adapted to spermatozoa to overcome the coenzyme-degrading activity of seminal plasma. 2. The content of total NAD+and NADH was determined in the spermatozoa of ram, bull, boar, stallion and cock. NADP+and NADPH were not detected in ram spermatozoa. 3. The oxidation state of sperm NAD depended on the seminal plasma, the removal of which produced a change in the percentage oxidation state of the coenzyme, 100×[NAD+/(NAD++NADH)], without altering the total content of NAD++NADH. 4. In suspensions of washed ram spermatozoa, incubated anaerobically at 25°C, the percentage oxidation state of NAD declined with increasing spermatozoa concentration. 5. When ram or boar spermatozoa that had been previously washed and resuspended in Ringer phosphate medium, were incubated anaerobically at 25°C with various substances, pronounced effects on the percentage oxidation state of NAD could be observed with l-lactate, pyruvate, oxaloacetate, dihydroxyacetone, formaldehyde and glyceraldehyde; sorbitol and acetoacetate acted only on ram spermatozoa; fructose, glucose, mannose and acetaldehyde acted predominantly on boar spermatozoa. Formaldehyde lowered the (NAD++NADH) content of ram spermatozoa, but none of the other substances had a comparable effect. 6. The percentage oxidation state of sperm NAD was not influenced by exogenous cysteine, cystine, ergothioneine or ascorbate. 7. A highly active sorbitol dehydrogenase could be prepared from ram, but not from boar, spermatozoa. 8. Sorbitol, acetoacetate and 3-hydroxybutyrate effectively supported the respiration of ram, but not boar, spermatozoa. 9. ‘Cold shock’, resulting from sudden cooling of spermatozoa, abolished motility completely and irreversibly but produced only a slow and partial decrease in the total NAD content. Slight over-heating, sufficient to produce loss of motility, had no adverse effect on the total NAD content. 10. Storage of ram sperm at 14°C produced only a small decrease of NAD after 2 days, but subsequently the loss became greater.