Pathways to maladaptive parenting with mothers and their conduct disordered children.

Maladaptive parenting with conduct disordered children appears to involve two social interaction traps called compliance and inconsistency. A mother's participation in these traps is thought to be influenced by her child, by the quality of the mother's life, and by her perceptions of child behavior. This study was an attempt to assess these influences and to examine their correlations with maternal trap participation. To do so, 33 clinic-referred conduct disordered children and their mothers were evaluated through direct observation, maternal self-reports, and maternal observations of home videotapes depicting mother and child. The data were then analyzed as correlational paths among trap measures and measures of the suspected influences. Results showed the two traps to be distinctly different processes, and inconsistency appeared to represent a more complex process than did compliance. Discussion of the findings centered on problems in maternal care of conduct disordered children and prospects for clinical interventions with these mother-child dyads.


Generalization in social behavior research with children and youth who have emotional or behavioral disorders.

This article presents a critical analysis of social behavior research with students who have emotional or behavioral disorders (EBD). In particular, it focuses on issues of generalized responding and a technology of generalization promotion. From a review of selected studies the conclusion is drawn that (a) researchers too rarely examine generalization in social behavior research, (b) studies that provide analysis of generalization rely on the least analytic approaches, and (c) generalization is seldom presented as a dependent variable of central interest in social behavior research with EBD students. Guidelines for further research in the area of generalization promotion are offered.


[Light and electron microscopic studies of calf kidneys after exposure to subtoxic lead levels].

Eight calves were fed from the fourth to the twentieth week of age a milk powder diet containing 40 mg lead acetate per kg dry substance. This is twice the lead concentration legally permitted. In average, each animal has daily taken up 0.834 mg lead per kg body weight. Eight calves were used as controls. The animals gained the usual fattening weights and did not show clinical symptoms. Beginning with week 14, increased levels of blood urea were encountered in the animals which received lead in their diet. Morphologically, severe renal lesions were found in these calves. The kidneys were increased in weight, pale and of firm consistency. Histologically, fibrosis and periglomerular interstitial non-purulent nephritis were found. Light- and electronmicroscopically, numerous intranuclear inclusion-bodies typical for lead poisoning were demonstrated in epithelial cells of proximal tubules. The results show, that daily uptake of less than 1 mg lead per kg body weight must be considered as a toxic dose for calves, and not 5-7 mg as stated in literature.


[Morphometric studies of the proximal tubules of the rat kidney considering the effect of the method of killing].

120 male rats belonging to two different strains (Han: WIST, Han: SPRD) and two different weight groups (group I: lighter body weight, group II heavier body weight) were killed on three different methods (decapitation, Nembutal -overdose greater than 250 mg/kg b.w. i.p. less than, exsanguination in Nembutal -anaesthesia greater than 100 mg/kg b.w. i.p.). Subsequently, changes in tubuli contorti I of the left kidneys were examined morphometrically. The mean total area, luminal area and epithelial area were evaluated as well as the mean diameter of the tubular nuclei. Concerning the areas of the proximal tubules only the luminal areas show differences between the Wistar and the Sprague-Dawley strain. The total, luminal and epithelial area in the rats belonging to the weight group II were significantly larger than in the rats belonging to weight group I. With one exception this could be noticed within both strains and all three modes of killing. In both strains and weight groups the rats were killed by Nembutal -overdose or exsanguination showed significantly smaller total, luminal and epithelial areas than the rats that were decapitated. Possible cause heretofore are discussed. Differences between the tubular areas of the rats that were killed by Nembutal -overdose and those that were killed by exsanguination after Nembutal -anaesthesia could not be established. The measurements of nuclei diameters in the proximal tubules did not lead to clearly different results between both strains, weight groups and the various modes of killing.


[Old age--a disease?].

The process of aging is discussed in relation to the environment of an individual and the evolution. Aging occurs hierarchically at a molecular, cellular, and systemic level. These physiologic processes of aging are accompanied by diseases, age-dependent and age-related diseases can be differentiated.


Peroxidase-antiperoxidase labelling of Leishmania amastigotes in formalin fixed and paraffin embedded tissue sections.

A modified PAP-Method (peroxidase-antiperoxidase) is established for laboratory use to demonstrate Leishmania spp. amastigotes in paraffin sections of experimentally and naturally infected dogs. The results demonstrate that the PAP technique is a valuable tool to detect Leishmania amastigotes in tissue sections with low parasite density.


[Formal pathogenesis, average age and breed distribution in the comparison of 61 Lysodren-treated and 36 untreated cases of canine hyperadrenocorticism which were dissected in the years 1975 to 1991 at the Institute for Veterinary Pathology of the Free University of Berlin].

61 Lysodren -treated dogs with hyperadrenocorticism are compared with 36 untreated dogs with the same disease. 75.14% of the treated dogs had pituitary-dependent-hyperadrenocorticism (PDH) whereas this diagnosis was found in 63.89% of the untreated dogs. A functioning adrenocortical tumor (FAT) was found in 13.11% of the treated dogs but was evident in 22.22% of the untreated dogs. Simultaneous pituitary and adrenal Cushing's syndrome (PDH/FAT) were found in 11.48% of the treated and 8.33% of the untreated cases. Sex distribution was equal in both groups. Overall slightly more females than males were affected by the disease. Poodles (38.14%) and dachshunds (23.71%) were the mainly affected races. Race-distribution of diseased animals is compared to the overall race-distribution in a ten years statistic (1981-1990) of all necropsied animals (Walter und Schwegler, 1992). The mean age of Lysodren -treated dogs with a PDH diagnosis (10.0 years) is lower as the mean age of untreated dogs with the same diagnosis (10.3 years). Reasons for this unexpected finding are discussed.


[Globular periodontal cement dysplasia in a black rhinoceros (Diceros bicornis)].

A globular periodontal cementous dysplasia in a 18 years old black rhinoceros (Diceros bicornis) is diagnosed by gross pathology, X-ray, and by histological examinations. The findings are discussed.


A mammalian epididymal protein with remarkable sequence similarity to snake venom haemorrhagic peptides.

Following spermatogenesis in the testis, mammalian spermatozoa pass into the epididymis, where they undergo changes which confer on them forward motility and the ability to recognize and penetrate the egg. Many of these maturation events involve androgen-regulated epididymal proteins which become associated with the sperm membrane, and/or effect changes to integral sperm membrane proteins. Here we report the sequence of an 89 kDa androgen-regulated protein from rat (Rattus norvegicus) and monkey (Macaca fascicularis) epididymis that is synthesized exclusively in the caput region and is localized on the apical surface of its principal epithelial cells. This protein shows remarkable similarity to a variety of proteases and disintegrins found in snake venoms and is similar to, but distinct from, the guinea-pig sperm surface PH-30 alpha/beta complex recently implicated in sperm-egg recognition and fusion.


Production, processing and partial purification of functional G protein beta gamma subunits in baculovirus-infected insect cells.

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.


Okadaic acid produces changes in phosphorylation and translocation of proteins and in intracellular calcium in human neutrophils. Relationship with the activation of the NADPH oxidase by different stimuli.

Okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, profoundly influenced the activity of the NADPH oxidase of human neutrophils. It strongly inhibited stimulation of superoxide generation by phorbol 12-myristate 13-acetate (PMA) and impaired translocation of protein kinase activity and of the two cytosolic components p47-phox and p67-phox to the plasma membrane. The increase in the phosphorylation of the cytochrome b-245 subunits p22-phox and gp91-phox after stimulation was also blocked. Inhibition of activity was associated with a decrease in cytosolic free Ca2+ and was reversed by the Ca2+ ionophore A23187, which also restored protein translocation and phosphorylation of the cytochrome. This effect of A23187 was itself blocked by preincubation with cyclosporin A, suggesting that calcineurin might be involved in the re-activation process. In contrast with PMA, the response to the bacterial peptide fMet-Leu-Phe was greatly prolonged after an initial decrease in the rate of onset of NADPH oxidase activity.


Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate.

The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.


Evidence that adhesion of electrically permeabilized platelets to collagen is mediated by guanine nucleotide regulatory proteins.

Adhesion of electrically permeabilized platelets to collagen was found to be essentially independent of free Ca2+ concentration in the medium. Addition of stable GTP analogues increased the proportion of adhering cells about 5-fold. This effect was inhibited by guanosine 5'-[beta-thio]diphosphate, cytochalasin D or monoclonal antibodies to glycoprotein Ia. In contrast, the protein kinase C inhibitor staurosporine had only a small effect on the GTP-analogue-enhanced adhesion of the permeabilized cells to collagen. These results suggest that a guanine nucleotide regulatory (G)-protein is directly linked to the collagen receptor and is involved in the actin-dependent recruitment of additional collagen receptors.


Immunoreactive folate-binding proteins from human saliva. Isolation and comparison of two distinct species.

Human saliva contains a single 72,000-M(r) species which specifically reacted with rabbit anti-[human placental folate receptor (PFR)] serum on SDS/PAGE and Western blots. Although a specific radioimmunoassay for human PFR and related folate-binding proteins (FBPs) identified 55 ng of cross-reacting material (CRM) per mg of crude salivary proteins, only a minor fraction (1.6 ng) specifically bound radiolabelled folate. The major fraction of CRM did not contain bound endogenous folate and did not bind radiolabelled folates. On the basis of folate binding, salivary CRM species to PFR were designated as either functional (f-FBP) or non-functional (nf-FBP) species respectively. nf-FBPs and f-FBPs were isolated by different purification schemes. Both purified f-FBPs and nf-FBPs migrated as a single apparent 72,000-M(r) species on SDS/PAGE, but on Sephacryl S-200 gel filtration and sucrose-density-gradient ultracentrifugation they were eluted/sedimented with 40,000-M(r) markers. Each microgram of purified f-FBP and nf-FBP was measured in the radioimmunoassay for PFR as being equivalent to 18 ng and 24 ng of CRM respectively, indicating low epitope-relatedness to PFR. The Kd of f-FBPs was 50 pM and 0.94 mol of folate was bound/mol of protein. f-FBPs exhibited an unusual dependence on Triton X-100 for optimal ligand binding, despite the fact that Triton X-100 micelle binding was not demonstrated. The relative order of affinity of f-FBPs for pteroylglutamate greater than methotrexate greater than 5-formyltetrahydrofolate greater than 5-methyltetrahydrofolate was also distinct from that of purified PFR. Whereas amino acid and carbohydrate analysis revealed that nf-FBP (M(r) 51,400) and f-FBP (M(r) 39,200) were distinct glycoproteins with 8 and 13% carbohydrate respectively, isoelectric focusing and immunological studies suggested some structural identity. The presence of f-FBP and nf-FBP in normal saliva raises new questions about their possible role in vivo.


Inhibition by heparin of Fe(II)-catalysed free-radical peroxidation of linolenic acid.

Heparin, in a concentration-dependent manner, inhibited the generation of conjugated dienes and thiobarbituric acid-positive substances when incubated with Fe2+ and gamma-linolenic acid. In the conjugated diene assay, other glycosaminoglycans, on a molar basis calculated with respect to their respective hydrated disaccharide repeat units, were less effective than heparin. Heparin which had been re-N-sulphonated after removal of both N-sulphonates and O-sulphates, and heparin in which iduronate residues had been reduced to idose residues, were largely unaffected in their activity. Removal of both N-sulphonates and O-sulphates greatly reduced the effectiveness of the heparin. Analysis of the effects of heparin fragments generated by heparinase I treatment of heparin showed that depolymerization decreased the effectiveness of the heparin. It is possible that heparins and related strongly acidic polysaccharides may function as endogenous antioxidants, and that sequestration by them, or harmless oxidation by them, of ions such as Fe2+, contributes to their effectiveness.


Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) beta-galactosidase.

Kinetic parameters for the hydrolysis of a series of deoxy and deoxyfluoro analogues of 2',4'-dinitrophenyl beta-D-galactopyranoside by Escherichia coli (lacZ) beta-galactosidase have been determined and rates found to be two to nine orders of magnitude lower than that for the parent compound. These large rate reductions result primarily from the loss of transition-state binding interactions due to the replacement of sugar hydroxy groups, and such interactions are estimated to contribute at least 16.7 kJ (4 kcal).mol-1 to binding at the 3, 4 and 6 positions and more than 33.5 kJ (8 kcal).mol-1 at the 2 position. The existence of a linear free-energy relationship between log(kcat./Km) for these compounds and the logarithm of the first-order rate constant for their spontaneous hydrolysis demonstrates that electronic effects are also important and provides direct evidence for oxocarbonium ion character in the enzymic transition state. A covalent intermediate which turns over only extremely slowly (t1/2 = 45 h) accumulates during hydrolysis of the 2-deoxyfluorogalactoside, and kinetic parameters for its formation have been determined. This intermediate is nonetheless catalytically competent, since it re-activates much more rapidly in the presence of the transglycosylation acceptors methanol or glucose, thereby providing support for the notion of a covalent intermediate during hydrolysis of the parent substrates.


Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several D-xylose isomerases.

D-Xylose isomerases are metal-ion (Mn2+, Co2+, Mg2+)-requiring tetrameric enzymes. Both the stoichiometry and the binding constants have been determined by titrating the metal-ion-free enzymes from five organisms (Actinomycetaceae and more divergent bacteria) with the respective metal ions using the enzyme activity as indicator of active complex-formation. The following characteristics have been observed for each specific isomerase: (i) two essential metal ion sites (one structural and one catalytic) exist per subunit; (ii) the metal ion binding at one site does not affect the binding at the other site; (iii) of the four possible configurations E, aE, Eb and aEb, only the double-occupied enzyme is active; (iv) the metal ion activation is a time-dependent process; (v) the dissociation constants for both the structural and catalytic sites may be identical or may differ by one or higher orders of magnitude; (vi) metal ion binding is stronger in the order Mn2+ greater than Co2+ much greater than Mg2+; (vii) pronounced increases in Km values concomitant with decreasing equivalents of metal ion added are only observed in the presence of Mg2+ ions.


Expression of ileal glucagon and peptide tyrosine-tyrosine genes. Response to inhibition of polyamine synthesis in the presence of massive small-bowel resection.

Massive small-bowel resection results in a marked adaptive response in the residual terminal ileum. Increased polyamine synthesis is a necessary component of this response. The ileal L-cell-derived peptides enteroglucagon and peptide tyrosine tyrosine (PYY) have been implicated as humoral mediators of this response. We have previously reported a rapid and sustained increase in glucagon mRNA concentrations after massive small-bowel resection. In this study using an inhibitor of the rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase, we have demonstrated that the response of the glucagon and PYY genes to massive small-bowel resection is dependent on polyamine biosynthesis. In addition, we have examined the response of both the ornithine decarboxylase and c-jun genes in this model of intestinal adaptation.


Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate.

The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.


Sphingomyelin content of intestinal cell membranes regulates cholesterol absorption. Evidence for pancreatic and intestinal cell sphingomyelinase activity.

Micellar cholesterol uptake and secretion were investigated in the human intestinal cell line CaCo-2 following depletion of apical membrane sphingomyelin. The addition of exogenous sphingomyelinase, which hydrolysed 60% of prelabelled sphingomyelin, resulted in a 50% decrease in the uptake of cholesterol from bile salt micelles. The flux of membrane cholesterol into the cell by the hydrolysis of membrane sphingomyelin decreased the rate of cholesterol synthesis by 43% and inhibited hydroxymethylglutaryl-CoA reductase activity by 54%. Moreover, the rate of cholesterol esterification was increased 4-fold. Total cellular cholesterol mass was unchanged by the addition of sphingomyelinase; however, cholesteryl esters increased by 50% and the amount of unesterified cholesterol decreased significantly. The basolateral secretion of cholesterol mass was also decreased following sphingomyelin hydrolysis. Human pancreatic juice was found to contain neutral sphingomyelinase activity which required taurocholate for full expression. The presence of neutral sphingomyelinase activity was also documented in membranes prepared from CaCo-2 cells and in whole homogenates from human duodenal biopsies. The data suggest that the amount of sphingomyelin present in the apical membrane of the intestinal absorptive cell regulates cholesterol uptake from bile salt micelles. Sphingomyelinase activity within intestinal cells and in pancreatic juice could alter the sphingomyelin content of brush-border membranes of small intestinal absorptive cells and thus regulate the amount of cholesterol absorbed by the gut.


Isolation and characterization of the promoter for the gene coding for the 68 kDa carnitine palmitoyltransferase from the rat.

Carnitine palmitoyltransferase (CPT) regulates the flux of long-chain fatty acids into the mitochondria for subsequent beta-oxidation. A 485 bp segment of the promoter for the gene encoding the 68 kDa CPT was isolated from a rat lambda DASH genomic library using the polymerase chain reaction. The promoter contained a consensus binding sequence for CREB (cyclic AMP response element binding protein) at -153 to -166, and for C/EBP alpha (CCAAT/enhancer binding protein) at -115 to -128. DNAase I footprinting using proteins isolated from rat liver nuclei indicated the presence of several regions of nuclear protein binding, most notably at -95 to -130, at -273 to -295, and at a wide region encompassing -395 to -465. DNAase I footprinting studies with purified CREB and C/EBP alpha confirmed that protein binding to DNA occurred at the sites predicted by the consensus sequences. The segment containing 481 bp of 5' flanking sequence plus 181 bp of untranslated mRNA was ligated to the structural gene for chloramphenicol acetyltransferase (CAT). When this plasmid was transfected into Hep G2 cells, CAT activity was stimulated 7-fold by addition of 1 mM-8-bromo-cyclic AMP (8-Br-cAMP) or co-transfection of the expression vector coding for the catalytic subunit of protein kinase A (PKA). The ability of several known second messengers and transcription factors to stimulate transcription of 68 kDa CPT promoter-CAT reporter was tested in co-transfection experiments. 68 kDa CPT promoter-CAT reporter transcription activity was stimulated 7-fold by addition of 8-Br-cAMP, and this induction was depressed 50% by the addition of phorbol esters. When the 68 kDa CPT promoter-CAT reporter was co-transfected with an expression vector for CREB or C/EBP alpha, transcription was increased 3- and 10-fold respectively. 8-Br-cAMP caused an additional 8-fold induction in the presence of each factor to yield 25- and 80-fold induction respectively. Co-transfection of the expression vector for c-jun also increased the CAT activity driven by the 68 kDa CPT promoter, while co-transfection with the expression vector for c-fos had no effect. When expression vectors for both c-jun and c-fos were co-transfected with the 68 kDa CPT promoter, c-fos depressed the induction seen with c-jun alone.


Quantification of the accumulation and degradation of beta-very-low-density lipoproteins in vivo using a 19F-containing residualizing label and n.m.r. spectroscopy.

beta-Very-low-density lipoproteins (beta-VLDL)O were conjugated to the 19F-containing residualizing label, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), to determine whether the metabolism of this lipoprotein fraction could be characterized in vivo with n.m.r. spectroscopy. Solution state 19F high-resolution n.m.r. spectroscopy of DLBA-beta-VLDL, containing either intact apoproteins or selectively enzymically digested products, demonstrated that the extent of degradation could be distinguished by differences in spin-spin relaxation times (T2 times). DLBA-beta-VLDL was injected intravenously into rabbits, and accumulation of 19F in hepatic tissue was quantified non-invasively by n.m.r. spectroscopy 5 and 30 h after injection. In addition to quantifying the accumulation of DLBA-beta-VLDL in hepatic tissue, a marked decrease (approx. 100 Hz) in the linewidth of 19F resonance from labelled lipoproteins was observed at 30 h compared with the 5 h interval in continuously monitored animals. The change in linewidth was consistent with a decrease in molecular size that occurred during protein degradation, resulting in increased T2 times. To demonstrate that T2 times can be used as an index to quantify apoprotein degradation in vivo, relaxation measurements were performed on livers excised 20 h after injection of DLBA-beta-VLDL into rabbits. Two molecular motional fractions were revealed by relaxation profiles representing either an intact or an extensively degraded form of apoprotein. The amplitudes of each component were compared with results from trichloroacetic acid precipitation of liver homogenates acquired from rabbits 20 h after injection of beta-VLDL labelled with the radioiodinated analogue of DLBA, dilactitol-125I-tyramine. The amount of degraded apoprotein determined by n.m.r. spectroscopy and acid precipitation was 68.6 +/- 7.0% and 58.7 +/- 7.5% (n = 4) respectively. The results of this study demonstrate that 19F n.m.r. spectroscopy can be used to define the temporal characteristics of the hepatic metabolism of lipoproteins in vivo by quantifying both the tissue-specific accumulation and extent of apoprotein degradation. The methodology developed offers promise for the non-invasive, sequential and longitudinal evaluation of lipoprotein metabolism in vivo.


Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation.

Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd approximately 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd approximately 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proportion of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.


Partial purification and characterization of the sodium-ion-coupled 5-hydroxytryptamine transporter of rat cerebral cortex.

A procedure for the extensive purification of the Na(+)-coupled 5-hydroxytryptamine transporter of rat cerebral cortex has been developed. The 5-hydroxytryptamine transporter was solubilized with the non-ionic detergent digitonin, and the detergent extracts were subjected to sequential affinity chromatography on a citalopram-based agarose support and wheat-germ-agglutinin-Sepharose. 5-Hydroxytryptamine transporters in the affinity-purified preparation were identified by using the selective 5-hydroxytryptamine-uptake inhibitor [3H]paroxetine, and were shown to display a similar pharmacological profile to those present in particulate preparations. An overall transporter purification of around 2000-fold was achieved with a 9% recovery. SDS/PAGE of affinity-chromatographed material starting from detergent extracts incubated in the presence or absence of 1 mM-citalopram indicated that a polypeptide of M(r) 73,000 corresponded to the 5-hydroxytryptamine-transporter protein.


Interaction between protein kinase C and Exo1 (14-3-3 protein) and its relevance to exocytosis in permeabilized adrenal chromaffin cells.

The roles of protein kinase C (PKC) and Exo1 in exocytosis from digitonin-permeabilized adrenal chromaffin cells were explored by using exogenous purified proteins in a run-down/reconstitution system. The stimulatory action of Exo1 on exocytosis from run-down cells was found to be completely dependent on the continuous presence of exogenous MgATP, suggesting that it acts on the slow phase of exocytosis [Holz, Bittner, Peppers, Senter & Eberhard (1989), J. Biol. Chem. 264, 5412-5419]. Partially purified rat brain PKC was found to be able to stimulate Ca(2+)-dependent exocytosis from run-down cells in a dose-dependent manner. This effect was indeed due to PKC and not a contaminant in the PKC fraction, since the PKC activator phorbol 12-myristate 13-acetate (PMA), under conditions in which control secretion was not affected, potentiated the effect of the exogenous PKC in stimulating secretion. Furthermore, although either PKC or Exo1 alone could stimulate exocytosis from run-down cells, the effect of combining the fractions was synergistic, as had previously been observed using PMA treatment combined with Exo1 incubation [Morgan & Burgoyne (1992) Nature (London) 355, 833-836]. The observed synergy between PKC and Exo1 was not due to PKC-mediated phosphorylation of Exo1, and Exo1 was found not to affect PKC activity in enzyme assays. We conclude that PKC and Exo1 act synergistically in the slow phase of Ca(2+)-dependent exocytosis from adrenal chromaffin cells. Furthermore, PKC does not directly affect Exo1, but rather enhances the activity of Exo1 by a putative phosphorylation of another, unidentified, component of the exocytotic machinery which facilitates the action of Exo1 in exocytosis.


Permeability of rat liver microsomal membrane to glucose 6-phosphate.

Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer revealed the following. (1) The increase in extravesicular osmolality by addition of glucose 6-phosphate or mannose 6-phosphate (25 mM each) caused a rapid shrinking of microsomal vesicles. After shrinkage, a rapid swelling phase (t1/2 approx. 22 s) was present with glucose 6-phosphate but absent with mannose 6-phosphate, indicating that the former had entered microsomal vesicles, but the latter had not. (2) Almost identical results were obtained in the absence of any glucose 6-phosphate hydrolysis, i.e. with microsomes pre-treated with 100 microM-vanadate. (3) The anion-channel blocker 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) suppressed the glucose 6-phosphate-induced swelling phase. (4) The swelling phase was more prolonged as the glucose 6-phosphate concentration increased (t1/2 = 16 +/- 3, 22 +/- 3 and 35 +/- 4 s with 25 mM, 37.5 mM- and 50 mM-glucose 6-phosphate respectively). The behaviour of glucose-6-phosphatase activity of intact and disrupted microsomes measured in the presence of high concentrations (less than 30 mM) of substrate also indicated the saturation of the glucose 6-phosphate permeation system by extravesicular concentrations of glucose 6-phosphate higher than 20-30 mM. Additional experiments showed that vanadate-treated microsomes pre-equilibrated with 0.1 mM- and 1.0 mM-glucose 6-phosphate (and [1-14C]glucose 6-phosphate as a tracer) rapidly (t1/2 less than 20 s) released [1-14C]glucose 6-phosphate when diluted in a glucose 6-phosphate-free medium. The efflux of [1-14C]glucose 6-phosphate was largely prevented by DIDS, allowing an evaluation of the intravesicular space of glucose 6-phosphate of approx. 1.0 microliter/mg of microsomal protein.


Partial characterization of natural and recombinant human soluble CD23.

The purification to homogeneity of an active soluble 25 kDa fragment of CD23, produced in insect cells using the baculovirus expression system, is described. Peptide mapping and analysis by Edman degradation and mass spectrometry permitted partial characterization of the protein. A total of 165 out of 172 residues, including N-terminal and C-terminal regions, were mapped. The positions of the two disulphide bonds in the IgE-binding region were also determined: residue 110 is joined to residue 124, and residue 42 to residue 133. Natural CD23 25 kDa fragment was also analysed and found to possess the same disulphide bond arrangement. These results extend the previously noted sequence similarity with lectins to elements of secondary structure.


Human interleukin-5 expressed in Escherichia coli has N-terminal modifications.

Recombinant human interleukin-5 exists as four major isoforms all possessing N-terminal methionine. Peptide mapping and subsequent analysis by fast-atom-bombardment mass spectrometry (f.a.b.-m.s.) have shown that N-terminal modifications are the cause of the charge heterogeneity. In order of decreasing abundance, these are unmodified methionine, retention of N-terminal formyl group, oxidation of N-terminal methionine to sulphoxide and carbamoylation of the N-terminus. These results were confirmed by analysis of the reduced and alkylated intact protein by electrospray-ionization mass spectrometry. The implications of these findings for the production and characterization of recombinant proteins are briefly discussed.


The activity and subcellular distribution of the peroxisomal enzyme acyl-CoA oxidase in human blood platelets.

The peroxisomal enzyme acyl-CoA oxidase is localized in the 'dense-tubular-system-enriched fraction', probably identical with the endoplasmic reticulum, in human blood platelets. This localization is strongly different from the localization of catalase which seems to be a cytosolic enzyme, in agreement with Marcus, Zucker-Franklin, Safir & Ullman [(1966) J. Clin. Invest. 45, 14-28]. A localization of acyl-CoA oxidase in the endoplasmic reticulum seems to be in good accordance with the important role of peroxisomes in the metabolism of prostaglandins, as recently demonstrated by Diczfalusy, Kase, Alexson & Bjørkhem [(1991) J. Clin. Invest. 88, 978-984].


Two distinct transcriptional activities of pea (Pisum sativum) chloroplasts share immunochemically related functional polypeptides.

An RNA polymerase activity has been purified from pea (Pisum sativum) chloroplast extracts with a distinct transcriptional specificity for a chloroplast messenger gene. This activity (ms-RNA pol) differs from the pea RNA polymerase preparation reported by Sun, Shapiro, Wu & Tewari [(1986) Plant Mol. Biol. 6, 429-439], which specifically transcribes only the rRNA gene (rb-RNA pol). The specificity of transcription has been assessed by the synthesis in vitro of discrete transcripts of predicted sizes using cloned promoter regions of the chloroplast psbA and 16 S rRNA genes. The ms-RNA pol preparation, with polypeptides ranging in apparent molecular mass from 22 to 180 kDa, correctly initiates transcription from recombinant plasmids containing the psbA promoter and does not support 16 S rRNA promoter-directed transcription. The two activities differ also in their response to Mn2+ ions. To investigate whether the two transcriptional activities share common functional polypeptides, monoclonal antibodies were developed against the rb-RNA pol preparation. Three clones were selected on the basis of their ability to inhibit transcription in vitro of the 16 S rRNA gene by rb-RNA pol. The antibodies from these clones independently recognized three polypeptides with molecular masses of 27, 90 and 95 kDa on immunoblots. Antibodies cross-reacting with the 90 kDa polypeptide completely eliminated the specific retardation of an end-labelled 16 S rRNA promoter fragment in a mobility-shift assay, whereas the antibodies against the 95 kDa polypeptide resulted in the formation of a ternary complex (enzyme-DNA-antibody). The antibodies cross-reacting with the 27 kDa polypeptide, however, did not alter the mobility of the retarded DNA-enzyme complex on the gel. These antibodies also inhibited transcription in vitro of the psbA gene by ms-RNA pol and recognized polypeptides of identical molecular masses in the ms-RNA pol. These results show that the three polypeptides are functional components of the chloroplast transcriptional complex and appear to be involved in the transcription of both rRNA and mRNA genes. Transcriptional specificity is probably conferred by ancillary transcription factor(s) which remain to be identified.


A cortical phosphoprotein ('PP63') sensitive to exocytosis triggering in Paramecium cells. Immunolocalization and quenched-flow correlation of time course of dephosphorylation with membrane fusion.

We had previously shown that a phosphoprotein of 63 kDa ('PP63') is rapidly and selectively dephosphorylated during synchronous (less than or equal to 1 s) trichocyst exocytosis in Paramecium cells and then rephosphorylated within less than or equal to 1 min [Zieseniss & Plattner (1985) J. Cell Biol. 101, 2028-2035]. Using a new quenched-flow device, we now find a strict correlation between PP63 dephosphorylation and the process of membrane fusion, both occurring within 80 ms. Uptake of 32P over 90 min, followed by exocytosis and rephosphorylation for 1 min, results in a rather selective phosphorylation of the dephosphorylated form, P63, to PP63. Solubilization by repeated freezing and thawing allows isolations of P63 and PP63. On isoelectric focusing autoradiograms they have pI values of 6.05, 5.95 (major spots), 5.85 and 5.75. All spots are sensitive to alkaline, but not to acidic, hydrolysis (except for the pI-6.05 spot). On two-dimensional-gel autoradiograms the most prominent spot, of pI 5.95, is most extensively de- and re-phosphorylated. This spot, from de- and re-phosphorylated samples, was used to produce monospecific antibodies. A cortical localization of PP63 was revealed by producing Western blots from isolated cell-surface fragments ('cortices') and by immunofluorescence labelling. We assume that both P63 and PP63 are attached to cortical structures, e.g. around trichocysts, though they are partly soluble. This localization and the strict correlation of PP63 dephosphorylation with exocytotic membrane fusion suggests a role in fusion regulation.


Effect of side-chain amide thionation on turnover of beta-lactam substrates by beta-lactamases. Further evidence on the question of side-chain hydrogen-bonding in catalysis.

Two side-chain-thionated beta-lactams, a penicillin and a cephalosporin, have been prepared and found to be not significantly poorer as substrates of typical serine (classes A and C) beta-lactamases than are their oxo analogues. This result is interpreted to mean that any hydrogen-bonding site on these enzymes for the beta-lactam side-chain amide carbonyl group must be flexible and is more likely to be a passive rather than active or essential feature of the active site. Previously, data from crystal structures and site-directed mutagenesis had suggested that the side chain of Asn-132 of class-A beta-lactamases, a component of the conserved SDN loop, forms a hydrogen bond with the side-chain carbonyl of the beta-lactam substrate and may provide significant transition-state stabilization during catalysis. The thionocephalosporin was also equally as good as its oxo analogue as a substrate of the class-B beta-lactamase II of Bacillus cereus and not significantly less effective as an inhibitor of the Streptomyces R61 DD-peptidase; a tight hydrogen-bond donor site for the beta-lactam side-chain amide is apparently not present in these enzymes either.


The metabolism of tetradecylthiopropionic acid, a 4-thia stearic acid, in the rat. In vivo and in vitro studies.

The metabolism of [1-14C]tetradecylthiopropionic acid (TTP), a 4-thia stearic acid, and its sulphoxide, [1-14C]texadecylsulphoxypropionic acid (TTP-SO), has been studied in intact rats, in isolated rat hepatocytes, and in rat liver mitochondria. Two pathways of oxidation (beta-oxidation and omega-oxidation) have been demonstrated. TTP is incorporated, in vivo, into tissue triacylglycerol and phospholipids, it is oxidized to CO2, and it is excreted in urine, mainly as carboxypropylsulphoxypropionic acid and a little as carboxymethylsulphoxypropionic acid. TTP-SO is metabolized, in vivo, more rapidly to the same two omega-oxidation products. In hepatocytes TTP is incorporated into triacylglycerol and phospholipids even more rapidly than stearic acid. It is recovered mainly in the 1-position of phosphatidylcholine. Some is oxidized to CO2 and acid-soluble products. TTP-SO is mainly omega-oxidized to the same metabolites as are found in urine. A small fraction is incorporated into phospholipids or oxidized to CO2. In isolated mitochondria [1-14C]TTP is converted into 14CO2, radioactive malonic semialdehyde, and addition products of malonic semialdehyde. In the presence of phenylhydrazine, malonic semialdehyde phenylhydrazone is the dominating product. In soluble extracts of mitochondria [1-14C]malonic semialdehyde is oxidized directly to 14CO2 in the presence of CoA and NAD+, probably by the (methyl)malonic acid semialdehyde dehydrogenase (EC 1.2.1.27).


A study of the stabilization of tetrahedral adducts by trypsin and delta-chymotrypsin.

delta-Chymotrypsin has been alkylated by 1-13C- and 2-13C-enriched tosylphenylalanylchloromethane. In the intact inhibitor derivative, signals due to the 1-13C- and 2-13C-enriched carbon atoms have chemical shifts which titrate from 55.10 to 59.50 p.p.m. and from 99.10 to 103.66 p.p.m. respectively with similar pKa values of 8.99 and 8.85 respectively. These signals are assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. An additional signal at 58.09 p.p.m. and at 204.85 p.p.m. in the 1-13C- and 2-13C-enzyme-inhibitor derivatives respectively does not titrate when the pH is changed and it is assigned to alkylated methionine-192. On denaturation/autolysis of the 1-13C-enriched enzyme-inhibitor derivative these signals associated with the intact inhibitor derivative are no longer detected, and a new signal, which titrates from 56.28 to 54.84 p.p.m. with a pKa of 5.26, is detected. The titration shift of this signal is assigned to the deprotonation of the imidazolium cation of alkylated histidine-57 in the denatured/autolysed enzyme-inhibitor derivative. Model compounds which form stable hydrates and hemiketals in aqueous solutions have been synthesized. By comparing the 13C titration shifts of these model compounds with those of the 13C enriched trypsin- and delta-chymotrypsin-inhibitor derivatives, we deduce that, in both of the intact enzyme-inhibitor derivatives, the zwitterionic tetrahedral adduct containing the imidazolium cation of histidine-57 and the hemiketal oxyanion predominates at alkaline pH values. It is estimated that in both the trypsin and delta-chymotrypsin-inhibitor derivatives the concentration of this zwitterionic tetrahedral adduct is 10,000-fold greater than it would be in water. We conclude that the pKa of the oxyanion of the hemiketal in the presence of the imidazolium cation of histidine-57 is 7.9 and 8.9 in the trypsin and delta-chymotrypsin-inhibitor derivatives respectively and that the pKa of the imidazolium cation of histidine-57 is greater than 7.9 and greater than 8.9 when the oxyanion is present as its conjugate acid, whereas, when the oxyanion is present, the pKa of the imidazolium cation is greater than 11 in both enzyme-inhibitor derivatives. We discuss how these enzymes preferentially stabilize zwitterionic tetrahedral adducts in the intact enzyme-inhibitor derivatives and how they could stabilize similar tetrahedral intermediates during catalysis. It is suggested that substrate binding could raise the pKa of the imidazolium cation of histidine-57 before tetrahedral-intermediate formation which would explain the enhanced nucleophilicity of the hydroxy group of serine-195.(ABSTRACT TRUNCATED AT 400 WORDS)


Rat epididymal luminal fluid acid beta-D-galactosidase optimally hydrolyses glycoprotein substrate at neutral pH.

Several glycosidases, purified and characterized from mammalian tissues, have been shown to be optimally active under acidic conditions when p-nitrophenyl (PNP) or 4-methylumbelliferyl glycosides are used as substrates. Although high levels of the glycosidases are present in the epididymal lumen, their physiological role remains uncertain. To be functional, the glycosidases are expected to be enzymatically active at or near the physiological pH of luminal fluid. In this report, we demonstrate that the rat epididymal luminal fluid beta-D-galactosidase, optimally active toward PNP beta-D-galactoside at pH 3.5, shows maximum activity towards a glycoprotein substrate ([Gal-3H]fetuin) at neutral pH. Several lines of evidence, including immunoprecipitation studies using antibody to the acid beta-D-galactosidase, and substrate competition studies, indicate that PNP galactosidase and [3H]Gal galactosidase activities are caused by a single enzyme, and that the two substrates are probably cleaved by the same catalytic site(s). Competition studies with various disaccharides indicate that this enzyme is capable of cleaving a variety of galactose linkages found in both O- and N-linked oligosaccharides. Molecular-sieve column chromatography of the beta-D-galactosidase of luminal fluid under several conditions of buffer and pH show that, whereas the enzyme eluted as a tetramer (apparent M(r) 320,000) under acidic conditions (pH 3.5-4.3), only dimers and monomers (apparent M(r) 180,000 and 92,000 respectively) were observed in neutral conditions (pH 6.8). This aggregation/dissociation phenomenon is reversible. These studies indicate that beta-D-galactosidase is present in the luminal fluid in dissociated forms, and is therefore optimally active towards glycoprotein substrates at physiological pH. The potential role of the enzyme in modification of sperm surface glycoproteins is discussed.


Turnover of glycogen phosphorylase in the pectoralis muscle of broiler and layer chickens.

Glycogen phosphorylase is a major sarcoplasmic protein in chicken pectoralis muscle, constituting approx. 4% of the total protein complement. In slow-growing layer chicks phosphorylase accumulated in parallel with muscle accretion, but in fast-growing broiler chicks the concentration of phosphorylase in the muscle increased (from 5 to 8 mg/g wet wt.) with time. In a 5-week period, the total amount of phosphorylase in the pectoralis muscles increased 18-fold in broiler chicks (from approx. 75 to 1400 mg total), but only 3-fold (from approx. 100 to 270 mg total) in layers. Pyridoxal phosphate, the cofactor of the enzyme glycogen phosphorylase, was used as a specific label to measure the rate of degradation of the enzyme in the pectoralis muscle of growing broiler and layer chickens in vivo. In young animals, the fractional rate of phosphorylase synthesis was similar in broiler and layer chickens (approx. 15%/day), but the rate of degradation in layers (5%/day) was 5-fold higher than in broilers (1%/day). As the animals aged, the rate of synthesis decreased, but more so in layers than in broilers. The rate of degradation of phosphorylase also decreased in layers, but in broilers it remained at the low level seen in young animals. The dramatically higher rate of phosphorylase accretion in the pectoralis muscles of the broilers is therefore achieved by an initial lower rate of degradation combined with a sustained difference between rates of synthesis and degradation.


Characterization of a human class-Theta glutathione S-transferase with activity towards 1-menaphthyl sulphate.

A purification scheme is described for a glutathione S-transferase (GST) from human liver that catalyses the conjugation of 1-menaphthyl sulphate (MS) with GSH; the method devised results in an approx. 500-fold increase in specific activity towards MS. The human enzyme which metabolizes MS is a homodimer comprising subunits of M(r) 25,100, and immunochemical experiments have shown it to be a member of the class-Theta GSTs. Automated Edman degradation of this enzyme has confirmed that it is a Theta-class GST bu the amino acid sequence obtained differs from that of GST theta described previously [Meyer, Coles, Pemble, Gilmore, Fraser & Ketterer (1991) Biochem. J. 274, 409-414]. We have therefore designated the enzyme that catalyses the conjugation of MS with GSH GST T2-2* (in the absence of complete amino acid sequence data, the T1 and T2 subunits are provisionally designated T1* and T2*); the evidence which indicates that GST theta (which should possibly now be called GST T1-1*) and GST T2-2* represent distinct isoenzymes is discussed.


Clearance from plasma of lymph chylomicrons and chylomicron remnants labelled with 125I-tyramine-cellobiose.

The aims of the present study were to evaluate the metabolism of chylomicrons (CM) and of CM remnants after labelling with radioactive iodine and converting the iodinated CM into remnants in vitro. Lymph CM were radiolabelled with 125I or sham-labelled with 127I by either the ICl procedure or the tyramine-cellobiose (TC) procedure, then injected into rats. The clearance from plasma of the iodinated CM was compared with control non-iodinated lipid-labelled CM. After iodination with ICl, the plasma removal of endogenously labelled CM was significantly different from non-iodinated CM, with increased uptake of CM triacylglycerols by the liver. In contrast, the clearances from plasma and the uptake by organs of radiolabelled lipids of CM iodinated by the TC method (TC-CM) were similar to control CM. About 40% of the label from 125I-TC-CM was insoluble in 50% propan-2-ol, indicating association with CM apolipoprotein B48. Only about 8% of label was lipid soluble, mostly in phosphatidylethanolamine. Radioactivity from 125I-TC-CM injected intravenously in rats was cleared rapidly and by 30 min only 20% remained in plasma, whereas 48% was recovered in the liver. After fractionation of the plasma by density-gradient ultracentrifugation, most label remained associated with d (relative density) less than 1.006 lipoproteins. In intact rats label was also found associated with the low-density and high-density lipoprotein fractions of plasma. When the liver was excluded from circulation, the recovery of label in low-density- and high-density-lipoprotein fractions was greatly decreased. CM remnants were prepared in vivo by injecting 125I-TC-CM into functionally hepatectomized donors and compared with remnants prepared in vitro by incubation with purified bovine milk lipoprotein lipase. Although remnants prepared in vitro cleared from plasma slower than remnants prepared in vivo, the size, lipid composition and apolipoprotein profile on gradient PAGE of the remnants were similar. We conclude that labelling of CM by the TC method avoided the 'artefactual' changes in metabolism seen after labelling by the ICl procedure. CM remnants when prepared in vitro using lipoprotein lipase were found to be similar to those prepared in vivo after injection into functionally hepatectomized rats.


Thrombin promotes actin polymerization in U937 human monocyte-macrophage cells. Analysis of the signalling mechanisms mediating actin polymerization.

The U937 human monocyte-macrophage cell line was used to examine the effect of thrombin, an ill-defined chemoattractant, on the polymerization of actin, a process essential for cell motility. In differentiated macrophage-like U937 cells, thrombin (0.5-50 units/ml) caused a rapid dose-dependent increase in the formation of filamentous (F-) actin, detected by the staining of F-actin with the fluorescent toxin, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In contrast with other chemoattractants such as N-formylmethionyl-leucylphenylalanine or C5a, actin polymerization in response to thrombin occurred via a pertussis-toxin-insensitive G1-(inhibitory G-protein) independent signalling pathway. Further, this response was not affected by the Ca2+ chelator EGTA or by the specific protein kinase C (PKC) inhibitor RO-31-8220. The response to thrombin was not mimicked by the Ca2+ ionophore ionomycin or by the direct PKC activator phorbol 12-myristate 13-acetate. The thrombin response was, however, inhibited by the non-specific protein kinase inhibitor staurosporine. The present results suggest that in U937 cells thrombin stimulates the formation of F-actin via a signalling pathway independent of (i) the activation of PKC, (ii) the mobilization of intracellular Ca2+ and (iii) the activation of Ca(2+)-dependent protein kinases, but dependent on the activation of an undefined staurosporine-sensitive protein kinase.


Kinetic and inhibition studies on catechol-O-methyltransferase affinity labelling by N-(3,4-dihydroxyphenyl)maleimide.

Initial velocity and product inhibition studies have been performed on soluble catechol-O-methyltransferase which has been partially purified from pig liver. The results are consistent with an ordered reaction mechanism, in which S-adenosyl-L-methionine (AdoMet) is the leading substrate. The enzyme is irreversibly inhibited by maleimide derivatives in a biphasic manner, which suggests a differential reaction with two thiol groups. N-(3,4-Dihydroxyphenyl)maleimide, which has a reactive moiety (maleimide ring) and an affinity moiety (catechol ring), acts as an affinity labelling compound on the more reactive SH group; AdoMet and Mg2+ protect against this modification. Total protection of this SH group results in a pseudo-first-order inhibition of the enzyme, with the apparent rate constant being proportional to the inhibitor concentration. All the other maleimide derivatives studied inhibited the enzyme by reacting with one of the two SH groups in a non-specific manner. The reaction of the other, more reactive, SH group was either specific (active-site-directed) or non-specific, depending on the substituent present in the affinity moiety and also on the length of an intermediate chain of methylene groups present between this moiety and the reactive maleimide ring. In the presence of both AdoMet and Mg2+, 3,5-dinitrocatechol, a reversible inhibitor of the enzyme which is competitive with respect to the catechol substrate, protects the enzyme from inactivation by any of the maleimide derivatives. The adducts of these maleimide derivatives formed with dithiothreitol inhibit the enzyme reversibly, showing inhibition patterns that are consistent with the mechanism deduced from the initial velocity and product inhibition studies.


A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line.

We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.


Identification and characterization of aminopeptidases from Aplysia californica.

Aminopeptidase activities were identified in extracts of kidney, ovotestis, head ganglia, heart and haemolymph of Aplysia californica. These enzyme preparations hydrolysed [3H][Leu]enkephalin at the Try-1-Gly-2 bond as determined by h.p.l.c. analysis of cleavage products. In all these tissues, enkephalin-degrading aminopeptidase activities were present both in membrane-bound and cytosolic fractions. The bivalent-cation-chelating agent, 1,10-phenanthroline, inhibited kidney membrane aminopeptidase activity with an IC50 of 30 microM, suggesting that this enzyme is a metalloproteinase. The aminopeptidase inhibitor amastatin was the most potent inhibitor of [Leu]enkephalin degradation (IC50 25 nM) by membrane-bound aminopeptidase, and bacitracin, bestatin and puromycin were about 100-1000 times less potent. In contrast with membrane-bound aminopeptidase, the cytosolic form is sensitive to puromycin. Angiotensin-converting enzyme inhibitor had no effect on [Leu]enkephalin degradation by kidney membranes, while the neutral endopeptidase inhibitors were poor inhibitors of the enzymes in this preparation. The Km values of the aminopeptidase in the kidney membranes and cytosolic fractions for the [Leu]enkephalin substrate were 2.4 and 7.4 microM respectively. The aminopeptidase present in the kidney membranes also hydrolysed endogenous Phe-Met-Arg-Phe-amide peptide at the Phe-1-Met-2 bond as well as synthetic alanine p-nitroanilide and leucine p-nitroanilide. When used in a competition assay, these substrates inhibited hydrolysis of [3H][Leu]enkephalin, suggesting that the same enzyme degraded all these substrates. Taken together, these results suggest that Aplysia tissues contain both a membrane-bound aminopeptidase related to the mammalian aminopeptidase N and a cytosolic puromycin-sensitive aminopeptidase.


Conformational stability of bovine alpha-crystallin. Evidence for a destabilizing effect of ascorbate.

Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.


Respiratory control and substrate effects in the working rat heart.

31P n.m.r. spectroscopy was used to measure the concentration of phosphates commonly proposed to control oxidative phosphorylation. The effect of loading conditions, beta-adrenergic stimulation and different substrates (acetate, pyruvate or glucose) was examined under steady-state conditions in the isolated working rat heart. Oxygen consumption and haemodynamic variables were monitored continuously. In response to a 2-fold increase in afterload, there were no significant changes in [ADP], [ATP]/[ADP], or [ATP]/[ADP][Pi]. In the presence of isoprenaline, these variables also tended not to change from afterload. However, isoprenaline, at identical perfusion pressures, consistently decreased the phosphorylation potential and [ATP]/[ADP], but had little effect on [ADP]. Substrates altered the phosphate metabolites in a manner independent of oxygen consumption, and had only minor effects on the relationship between phosphates and work, in contrast with other studies. Thus, metabolites of ATP synthesis are not normally involved in respiratory control. The 31P n.m.r. spectrum can vary greatly, but does not predict oxygen consumption in this preparation. Substrates have no effect on the mechanism of respiratory control. Thus the normal control of respiration in the heart at steady state cannot occur at the level of its substrates. Rather, there must be concerted regulation of the numerous pathways, involving allostery and covalent modification. The attention of future research should be shifted away from the metabolites of ATP and towards identifying the effectors of such regulation.


Substrate specificity of the purine-2'-deoxyribonucleosidase of Crithidia luciliae.

The purine-2'-deoxyribonucleosidase of Crithidia luciliae catalyses an efficient deoxyribosyl transfer between a variety of purine bases, benzimidazole and 5,6-dimethylbenzimidazole. Since the deoxyriboside of a deoxyribosyl acceptor is necessarily also a substrate, the trans-N-deoxyribosylase activity of the enzyme allows a study of its specificity to be extended to a large number of purines and purine analogues. Amongst 27 different deoxyribosyl acceptors, only hypoxanthine gave rise to isomeric products. The introduction of methyl groups at appropriate positions in either purine or benzimidazole lowered the Michaelis constant, KB, for deoxyribosyl acceptors: by about 10-fold for 6-methylpurine (KB 351 +/- 87 microM) compared with purine (KB 3.91 +/- 0.8 mM) and by about 10(3)-fold for 5,6-dimethylbenzimidazole (KB 7.0 +/- 0.79 microM) compared with benzimidazole (Km,app. 7.8 +/- 2.4 mM). The maximal rates of deoxyribosyl transfer to different acceptors, on the other hand, varied by only 4.5-fold, and can be ascribed to decreases in the rate of release of the newly formed purine deoxyriboside from the enzyme.


Release of cell surface proteoglycans from differentiating colon cells proceeds by cleavage of lipophilic anchor peptides.

Heparan sulphate proteoglycans are rapidly released from VACO 10MS colon cancer cells that are triggered with phorbol esters to undergo terminal differentiation. This lag-free temperature-sensitive process is correlated with a conversion of the lipophilic proteoglycans of the cell surface into non-lipophilic proteoglycans that accumulate in the culture medium. The released proteoglycans are very similar to their lipophilic precursors in size, buoyant density and glycosaminoglycan characteristics; however, they exhibit slightly smaller core proteins after chemical and enzymic deglycosylation. The lipophilicity of the larger-sized core proteins of the cell-associated proteoglycans is also correlated with the presence of an easily iodinatable domain; this domain is missing in the released proteoglycans. Exogenous proteases (i.e. chymotrypsin, V8, trypsin and proteinase K) readily cleave this segment from the larger protease-resistant region of the proteoglycan structure. It is also released intact by treatment of the isolated proteoglycans with methanolic HCl. This component appears to be peptide in character, in that proteases readily degrade it and release iodotyrosines when the precursor has been iodinated. No evidence for the presence of covalently attached fatty acids in the cell-associated proteoglycans was found. These results are consistent with the hypothesis that the altered proteoglycan metabolism that is associated with the phorbol-ester-induced terminal differentiation of certain human colon cancer cells ensues upon the activation of a membrane-localized protease that cleaves a lipophilic anchor segment from the cell surface proteoglycans.


Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.).

The major b-type cytochrome in microsomal membrane preparations from developing endosperm of castor bean (Ricinus communis) was cytochrome b5. Cytochrome P-450 was also present. The microsomal membranes had delta 12-hydroxylase activity and catalysed the NAD(P)H-dependent hydroxylation of oleate to yield ricinoleic acid. CO had no effect on the hydroxylase activity. Rabbit polyclonal antibodies were raised against the hydrophilic cytochrome b5 fragment purified from cauliflower (Brassica oleracea) floret microsomes. The anti-(cytochrome b5) IgG inhibited delta 12-hydroxylase, delta 12-desaturase and cytochrome c reductase activity in the microsomes. The results indicate that electrons from NAD(P)H were transferred to the site of hydroxylation via cytochrome b5 and that cytochrome P-450 was not involved.


Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes.

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear 'mixed inhibition' pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.


ATPase-inhibitor proteins of brown-adipose-tissue mitochondria from warm- and cold-acclimated rats.

1. A group of male Sprague-Dawley rats (5-6 weeks old) was cold-acclimated at 4 degrees C for 4 weeks. Warm-acclimated controls remained at 24 degrees C. Total protein content of brown adipose tissue (BAT) increased more than 3-fold and total uncoupling protein (UCP) content increased more than 6-fold upon cold-acclimation. The concentration of UCP in isolated BAT mitochondria almost doubled. 2. Specific ATPase activity of the non-thermogenic BAT mitochondria (from warm-acclimated controls) was low and increased about 6-fold on addition of 1 microM-Ca2+, which raised free Ca2+ levels (measured by Fura-2) in the incubation media from 1.32 +/- 0.28 microM (mean +/- S.E.M.) to 2.29 +/- 0.39 microM [at which the Ca(2+)-binding ATPase-inhibitor protein (CaBI) is inactivated]. Correspondingly, the specific ATP synthetase activity of the non-thermogenic BAT mitochondria was high and was decreased by 74% by addition of 1 microM-Ca2+. 3. In contrast, specific ATPase activity of thermogenic BAT mitochondria (from cold-acclimated rats) was 5 times that of the control group, and addition of Ca2+ had only a small stimulatory response. Correspondingly, the specific ATP synthetase activity of the thermogenic BAT mitochondria was low, and the decrease by Ca2+ was small, albeit significant. 4. Extracts of BAT mitochondria from both groups of animals contained significant amounts of the ATPase-inhibitor protein of Pullman and Monroy (PMI) as well as of CaBI, as shown by gel electrophoresis. Kinetic studies of inhibition of mitochondrial ATPase activity showed that PMI activity was unaltered in extracts from the thermogenic BAT mitochondria, whereas CaBI activity was slightly but significantly increased. 5. The presence of active ATPase-inhibitor proteins in BAT mitochondria was shown for the first time. We conclude that uncoupling of oxidative phosphorylation occurs in thermogenic BAT mitochondria, even in the presence of the ATPase-inhibitor proteins.


Prokaryotic triterpenoids: O-alpha-D-glucuronopyranosyl bacteriohopanetetrol, a novel hopanoid from the bacterium Rhodospirillum rubrum.

A novel hopanoid bearing a glucuronopyranosyl residue linked via an alpha-glycosidic bond to the hydroxyl group of C-35 in bacteriohopanetetrol was isolated, from the type strain of Rhodospirillum rubrum as well as from a mutant lacking blue carotenoid.


Gene expression of calpains and their specific endogenous inhibitor, calpastatin, in skeletal muscle of fed and fasted rabbits.

To investigate the role of calpains in myofibrillar protein degradation in skeletal muscle and the regulation of their activity in vivo, we studied the effects of fasting on gene expression of calpains and calpastatin in the skeletal muscle of rabbits. In response to fasting, myofibrillar protein degradation increased 2-fold and mRNA levels of calpain I, calpain II and calpastatin were also increased. However, calpain and calpastatin activities remained unchanged. To investigate this discrepancy, we analysed polysomal calpain mRNA. Results indicated that fasting caused a 2-fold increase in the loading of calpain I and II mRNAs on ribosomes. Thus transcription of genes encoding calpain may be increased during fasting to ensure adequate synthesis of the proteinases needed to mobilize muscle protein reserves. The effect of fasting on calpain and calpastatin mRNA expression is shared by cathepsin D and proteasome C2 but not by beta-actin, implying that fasting invokes control of several proteolytic systems in skeletal muscle and underscores the possibility that each proteolytic system plays a role in the adaptation of skeletal muscle to the fasted state.


Small-angle X-ray-scattering studies of the C hordeins of barley (Hordeum vulgare).

Small angle X-ray scattering was used to study the solution conformation of the C hordeins of barley (Hordeum vulgare), a group of proteins whose primary structure consists predominantly of an octapeptide repeat motif. Measurements on the protein in 0.1 M-acetic acid at 25 degrees C are consistent with a model for the protein conformation of a stiff coil, the so-called 'worm-like' chain. The characteristic parameters (the Kuhn statistical segment length and the contour length) of the protein were calculated as 5.11 and 71.5 nm respectively.


The predominant calcimedins from Trypanosoma brucei comprise a family of flagellar EF-hand calcium-binding proteins.

The cellular complement of calcimedins was identified in Trypanosoma brucei by Ca(2+)-dependent association with phenyl-Sepharose. Predominant calcimedins with molecular mass of 23-26 kDa and 44 kDa, along with minor calcimedins of 96, 120 and 230 kDa, were obtained. The trypanosome calcimedins were unrelated to vertebrate annexins, based upon antibody cross-reactivity and an inability to associate in a Ca(2+)-dependent way with phospholipid vesicles comprised of phosphatidylserine or phosphatidylethanolamine/phosphatidylcholine (1:1, w/w). Partial sequence analysis demonstrated that 44 kDa calcimedin (Tb-44) contained an EF-hand calcium-binding loop. Five CNBr/tryptic fragments exhibited a total of 93% similarity with Tb-17, a 23 kDa EF-hand protein in T. brucei. The trypanosome calcimedins appeared to comprise a family of proteins, based on sequence similarities and antibody cross-reactivity of affinity-purified anti-Tb44 with the 23-26 kDa cluster. No evidence was found for Tb-44 in the related species T. cruzi, Leishmania taraentolae or Crithidia fasciculata. Antibodies against Tb-44 were localized by immunofluorescence along the flagellum of T. brucei. Immunoblot analysis of flagella-enriched preparations demonstrated that Tb-44 and the 23-26 kDa cluster were present in this structure. We conclude that annexin family members are not among the predominant trypanosome proteins that associate with phenyl-Sepharose in a Ca(2+)-dependent way. Instead, the major trypanosome calcimedins comprise a family of flagellar EF-hand calcium-binding proteins.


Structurally and functionally conserved regions of cytochrome P-450 reductase as targets for DNA amplification by the polymerase chain reaction. Cloning and nucleotide sequence of the Schizosaccharomyces pombe cDNA.

1. Alignments of the available cytochrome P-450 reductase amino acid sequences, and comparison with the crystal structure of ferredoxin-NADP reductase, indicate that two highly conserved regions are of functional importance. 2. Degenerate oligonucleotide primers, based on these sequences, were used in the polymerase chain reaction to amplify a 309 bp fragment of the cytochrome P-450 reductase gene from Schizosaccharomyces pombe for use as an homologous probe. 3. A 2.6 kb cDNA was cloned from a lambda library, and sequencing revealed an open-reading frame of 2034 bp encoding a protein of M(r) 76774. This protein shares 38-41% identity with other eukaryotic cytochrome P-450 reductases, and 30% identity with that of Bacillus megaterium. 4. Comparison of the N-terminal FMN-binding domain with flavodoxin, and the C-terminal FAD- and NADP-binding domain with ferredoxin-NADP reductase, indicates the presence of several functionally conserved regions. 5. The Sc. pombe cytochrome P-450 reductase gene was shown to contain no introns.


Biosynthesis of heparin. The D-glucuronosyl- and N-acetyl-D-glucosaminyltransferase reactions and their relation to polymer modification.

Oligosaccharides with the general structure [GlcA-GlcNAc]n-GlcA-aMan (aMan is 2,5-anhydro-D-mannose), derived from the Escherichia coli K5 capsular polysaccharide, were found to serve as monosaccharide acceptors for a GlcNAc-transferase, solubilized from a mouse mastocytoma microsomal fraction and implicated in heparin biosynthesis. Digestion of these oligosaccharides with beta-D-glucuronidase yielded acceptors for the GlcA-transferase that acts in concert with the GlcNAc-transferase. Assays based on the oligosaccharide acceptors showed broad pH optima for the two enzymes, centred around pH 6.5 for the GlcNAc-transferase and around pH 7.0 for the GlcA-transferase. The GlcNAc-transferase showed an absolute requirement for Mn2+, whereas the GlcA-transferase was stimulated by Ca2+ and Mg2+ but not by Mn2+. The GlcNAc acceptor ability of the [GlcA-GlcNAc]n-GlcA-aMan oligosaccharides increased with increasing chain length, as reflected by the apparent Km, which was 60 microM for a hexasaccharide but 6 microM for a hexadecasaccharide. By contrast, the Km for [GlcNAc-GlcA]n-aMan oligosaccharides in the GlcA-transferase reaction was higher, approximately 0.5 mM, and unaffected by acceptor size. After chemical modification of the oligosaccharides to obtain mixed N-substituents (N-unsubstituted, N-acetylated or N-sulphated GlcN residues), GlcNAc transfer was found to be virtually independent of the N-substituent pattern of the acceptor sequence. The GlcA-transferase, on the other hand, showed marked preference for an acceptor with a non-reducing-terminal GlcNAc-GlcA-GlcNSO3- sequence, which would thus have a lower Km for the enzyme than the corresponding fully N-acetylated structure. These results, along with our previous finding that chain elongation in a mastocytoma microsomal system is strongly promoted by concomitant N-sulphation of the nascent chain [Lidholt, Kjellén & Lindahl (1989) Biochem. J. 261, 999-1007], raise the possibility that the glycosyltransferases and the N-deacetylase/N-sulphotransferase act in concert during chain elongation, assembled into an enzyme complex.


Further characterization of the N-terminal copper(II)- and nickel(II)-binding motif of proteins. Studies of metal binding to chicken serum albumin and the native sequence peptide.

We have investigated the Cu(II)- and Ni(II)-binding properties of chicken serum albumin (CSA) and of the native sequence tripeptide derived from the N-terminus of this protein. Spectrophotometric and equilibrium dialysis experiments demonstrate that Cu(II) and Ni(II) bind non-specifically at the N-terminus of CSA. Proton displacement studies show that the histidine residue in the fourth position of the protein does not appear to participate in the binding of the two metals. Consistent results were obtained with the native sequence tripeptide L-aspartyl-L-alanyl-L-glutamic acid N-methylamide. The results presented here demonstrate that neither the glutamic acid residue in the third position nor the histidine in the fourth position participate in the binding of Cu(II) and Ni(II) to CSA. It is known, however, that a number of other albumins with a histidine residue in the third position possess high-affinity Cu(II)- and Ni(II)-binding sites. Our results provide further evidence that the N-terminal Cu(II)/Ni(II)-binding motif requires a histidine at the third position in order to bind Cu(II) and Ni(II) specifically.


Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen expression.

The Rh blood-group antigens are associated with human erythrocyte membrane proteins of approx. 30 kDa (the Rh30 polypeptides). Heterogeneously glycosylated membrane proteins of 50 and 45 kDa (the Rh50 glycoproteins) are coprecipitated with the Rh30 polypeptides on immunoprecipitation with anti-Rh-specific mono- and poly-clonal antibodies. We have isolated cDNA clones representing a member of the Rh50 glycoprotein family (the Rh50A glycoprotein). We used PCR with degenerate primers based on the N-terminal amino acid sequence of the Rh50 glycoproteins and human genomic DNA as a template and cloned and sequenced three types of PCR product of the expected size. Two of these products, Rh50A and Rh50B, gave the same translated amino acid sequence which corresponded to the expected Rh50 glycoprotein sequence but had only 75% DNA sequence similarity. The third product (Rh50C) contained a single base deletion, and the translated amino acid sequence contained an in-frame stop codon. We have isolated cDNA clones containing the full coding sequence of the Rh50A glycoprotein. This sequence predicts that it is a 409-amino acid N-glycosylated membrane protein with up to 12 transmembrane domains. The Rh50A glycoprotein shows clear similarity to the Rh30A protein in both amino acid sequence and predicted topology. Our results are consistent with the Rh30 and Rh50 groups of proteins being different subunits of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane. We mapped the Rh50A gene to human chromosome 6p21-qter, showing that genetic differences in the Rh30 rather than the Rh50 genes specify the major polymorphic forms of the Rh antigens.


Phospholipid molecular species composition of mouse liver nuclei. Influence of dietary n-3 fatty acid ethyl esters.

The effect of dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl esters on the individual molecular species composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was determined in mouse liver nuclei. After a 10 day feeding period, there was a depletion of the sn-2 position of n-6 polyunsaturated fatty acids (PUFA) and substitution with n-3 PUFA. EPA feeding significantly increased (P less than 0.05) diacyl PC and PE 16:0-20:5, n-3, 16:0-22:6,n-3, 18:0-20:5,n-3 and 18:0-22:6,n-3 relative to control (safflower oil ethyl ester fed) animals. In comparison, DHA feeding significantly increased (P less than 0.05) 22:6 n-3-containing species, specifically 18:1-22:6,n-3, 16:0-22:6,n-3 and 18:0-22:6,n-3 in PC, and 18:1-22:6,n-3, 16:0-22:6,n-3 and 18:0-22:6,n-3 in PE. In addition, the presence of 18:0-20:5,n-3 PC in the nuclei of DHA-fed rats and of 18:2-20:5,n-3, 18:1-20:5,n-3 and 18:0-20:5,n-3 in nuclear PE indicate that incorporation of DHA retroconversion (22:6,n-3-->20:5,n-3) products. These results indicate both EPA and DHA are extensively incorporated into nuclear phospholipids, and therefore could potentially influence gene function.


Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family.

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.


Carbachol stimulation of phospholipase A2 and insulin secretion in pancreatic islets.

Arachidonic acid has been implicated as a second messenger in insulin secretion by islets of Langerhans. D-Glucose, the major physiological stimulus, increases unesterified arachidonate accumulation in islets. We now show, for the first time, that the muscarinic agonist carbachol, at concentrations which stimulate insulin secretion, causes a rapid and nearly 3-fold increase in arachidonic acid accumulation in islets. The combination of glucose and carbachol has an additive effect on unesterified arachidonate release. There is a large component of secretagogue-induced arachidonate accumulation that is independent of extracellular Ca2+. Carbachol stimulation of arachidonic acid release is mediated by activation of phospholipase A2, as demonstrated by early increases in endogenous lysophosphatidylcholine. In addition to phospholipase A2 activation, carbachol-induced arachidonic acid accumulation also appears to involve diacylglycerol hydrolysis, since the diacylglycerol lipase inhibitor RG80267 partly inhibited arachidonic acid accumulation. In contrast, glucose-induced arachidonic acid accumulation appears to reflect diacylglycerol hydrolysis entirely. Our observations indicate that phospholipase A2 has an important role in muscarinic-induced insulin secretion.


Characterization of the human properdin gene.

A cosmid clone containing the complete coding sequence of the human properdin gene has been characterized. The gene is located at one end of the approximately 40 kb cosmid insert and approximately 8.2 kb of the sequence data have been obtained from this region. Two discrepancies with the published cDNA sequence [Nolan, Schwaeble, Kaluz, Dierich & Reid (1991) Eur. J. Immunol. 21, 771-776] have been resolved. Properdin has previously been described as a modular protein, with the majority of its sequence composed of six tandem repeats of a sequence motif of approximately 60 amino acids which is related to the type-I repeat sequence (TSR), initially described in thrombospondin [Lawler & Hynes (1986) J. Cell Biol. 103, 1635-1648; Goundis & Reid (1988), Nature (London) 335, 82-85]. Analysis of the genomic sequence data indicates that the human properdin gene is organized into ten exons which span approximately 6 kb of the genome. TSRs 2-5 are coded for by discrete, symmetrical exons (phase 1-1), which supports the hypothesis that modular proteins evolved by a process involving exon shuffling. TSR1 is also coded for by a discrete exon, but the boundaries are asymmetrical (phase 2-1). The sequence coding for the sixth TSR is split across the final two exons of the gene with the first 38 amino acids of the repeat coded for by an asymmetric exon (phase 1-2). This split at the genomic level has been shown, by alignment analysis, to be reflected at the protein level with the division of repeat 6 into TSR-like and TSR-unlike sequences.


cDNA cloning and sequence analysis of human pancreatic procarboxypeptidase A1.

Using polyclonal antibodies raised against human pancreatic procarboxypeptidases, a full-length cDNA coding for an A-type proenzyme was isolated from a lambda gt11 human pancreatic library. This cDNA contains standard 3' and 5' flanking regions, a poly(A)+ tail and a central region of 1260 nucleotides coding for a protein of 419 amino acids. On the basis of sequence comparisons, the human protein was classified as a procarboxypeptidase A1 which is very similar to the previously described A1 forms from rat and bovine pancreatic glands. The presence of the amino acid sequences assumed to be of importance for the zymogen inhibition by its activation segment, primarily on the basis of the recently reported crystal structure of the B form, further supports the proposed classification.


Structure of the bovine ETB endothelin receptor gene.

The structure of the gene encoding the bovine type B endothelin receptor (ETB) has been established and compared with those of other heptahelical receptors. The gene is present as a single copy in the bovine genome, as demonstrated by Southern blot analysis, and spans at least 36 kb. The coding region is divided into 7 exons separated by 6 introns, one of which is more than 23 kb in length. The exons correspond well to the structural domains of the receptor: the first exon encodes the first and second transmembrane domains, and each of the following transmembrane domains is encoded by a separate exon. The portion of the ETB protein sequence encoded by exon 3 is quite different from the corresponding ETA sequence, suggesting that this region is responsible for the distinct ligand specificities of the two receptor subtypes. The second intron interrupts the canonical Asp-Arg-Tyr sequence, which is located at the end of the third transmembrane domain of the heptahelical receptors, as with the substance P, substance K, dopamine D2 and dopamine D3 receptor genes. To map the 5' region of the gene and determine the start of transcription, primer-extended cDNAs were cloned and sequenced: multiple start sites were deduced with no apparent TATA box in the expected upstream region. Similar results were obtained by ribonuclease protection analysis.


Stimulation of phosphatidate synthesis in endothelial cells in response to P2-receptor activation. Evidence for phospholipase C and phospholipase D involvement, phosphatidate and diacylglycerol interconversion and the role of protein kinase C.

To investigate the stimulation of phosphatidic acid formation in bovine aortic endothelial cells by P2-purinergic agonists, we labelled AG4762 cells with [32P]P1 and stimulated in the presence of butanol. Under these conditions phospholipase D generated [32P]phosphatidylbutanol, whereas the [32P]phosphatidic acid from phospholipase C and diacylglycerol kinase was unchanged. The action of various purinergic agonists on both [32P]phosphatidic acid and [32P]phosphatidylbutanol was consistent with the presence of a P2Y receptor. The stimulation of phospholipase D was dependent on extracellular Ca2+ and was mostly transient (completed within 3 min), whereas the initial stimulation of phospholipase C was independent of extracellular Ca2+, followed by a Ca(2+)-dependent phase. The agonist stimulation of phospholipase D was dependent on protein kinase C, as judged by its sensitivity to the relatively selective protein kinase C inhibitor Ro 31-8220. These results show that purinergic-receptor-mediated stimulation of phosphatidic acid has three phases: an initial Ca(2+)-independent stimulation of phospholipase C, an early but transient Ca(2+)- and protein kinase C-dependent stimulation of phospholipase D, and a sustained Ca(2+)-dependent stimulation of phospholipase C. Using propranolol to inhibit phosphatidate phosphohydrolase, we provide evidence that phosphatidic acid derived from purinergic-receptor-mediated stimulation of the phospholipase C/diacylglycerol kinase route can itself be converted back into diacylglycerol.


Bovine colostrum CMP-NeuAc:Gal beta(1-->4)GlcNAc-R alpha(2-->6)-sialyltransferase is involved in the synthesis of the terminal NeuAc alpha(2-->6)GalNAc beta(1-->4)GlcNAc sequence occurring on N-linked glycans of bovine milk glycoproteins.

Bovine colostrum CMP-NeuAc:Gal beta(-->4)GlcNAc-R alpha(2-->6)-sialyltransferase (alpha 6-sialyltransferase) appears to be capable of catalysing alpha 6-sialylation of the disaccharide GalNAc beta(1-->4)GlcNAc to yield the trisaccharide NeuAc alpha(2-->6)GalNAc beta(1-->4)GlcNAc. This provides an enzymic basis for the occurrence of this sialylated structure on the N-linked glycans of a number of bovine milk glycoproteins. Competition experiments using Gal beta(1-->4)GlcNAc and GalNAc beta(-->4)GlcNAc as acceptors indicate that both substrates are recognized by a single active site on the alpha 6-sialyltransferase. Extrapolation of these results suggests that the NeuAc alpha(2-->6)GalNAc beta(1-->4)GlcNAc structural element occurring on the N-linked glycans of several human glycoproteins are similarly synthesized by the action of a Gal beta(1-->4)GlcNAc-R alpha(2-->6)-sialyltransferase.


Analysis and elimination of protein perturbation in infrared difference spectra of acyl-chymotrypsin ester carbonyl groups by using 13C isotopic substitution.

I.r. spectroscopy has been applied to the study of hydrogen-bonding of the unique ester carbonyl group of acylchymotrypsins in the oxyanion hole of the enzyme. This catalytic device provides electrophilic stabilization of negative charge in the transition states and tetrahedral intermediates along the reaction pathway. The use of 13C isotope substitution of the ester carbonyl group reinforces the previous observation [White & Wharton (1990) Biochem. J. 270, 627-637] that the ester carbonyl group is significantly polarized in the ground state by hydrogen bonding in the oxyanion hole. I.r. difference spectra of [carbonyl-12C]-minus [carbonyl-13C]-cinnamoyl-chymotrypsin as well as each of these acylenzymes minus free enzyme are reported. These spectra show that the contribution of protein perturbation (i.e. spectral features that arise from the enzyme which is distorted on acylation) in [carbonyl-12C]cinnamoyl-chymotrypsin minus free enzyme spectra is significant. The contribution of the perturbation components of the spectra is pH-dependent and can represent up to 50% of the total absorbance in the spectral region from 1690 to 1740 cm-1. Use of the isotopic difference method has allowed problems associated with protein perturbation to be eliminated. Similar difference spectra are presented for dihydrocinnamoyl-chymotrypsin. In this case the effect of perturbation is very marked and leads to the cancellation of the band assigned to the non-bonded conformation of the acyl group which has previously only been observed at higher pH. The isotopic difference method again proves reliable and shows that the frequency difference previously used to calculate the ground-state electronic strain induced by the oxyanion-hole catalytic device is not affected by the perturbation, although the amplitudes of the spectral features are different. A study of the deacylation of cinnamoyl-chymotrypsin in water and deuterium oxide using both u.v. and i.r. spectroscopies has confirmed that the use of deuterium oxide as solvent has no serious effect on the deacylation behaviour of the enzyme. I.r. bands assigned to nonproductive and productive conformers decline identically during deacylation, which shows that the conformers are in dynamic exchange on the reaction time-scale.


Characterization and identification of an epidermal-growth-factor-activated phospholipase A2.

The production of arachidonic acid (AA), which is involved in mitogenic signalling by epidermal growth factor (EGF), is most directly accomplished by the action of phospholipase A2 (PLA2). We demonstrate that EGF treatment of intact NEF cells rapidly activates a cytosolic PLA2, as measured in cell-free extracts by the release of radiolabelled AA from exogenously added 1-stearoyl-2-[1-14C]arachidonoyl phosphatidylcholine. Activation of PLA2 by EGF resulted in an enhanced Vmax. and no change in Km. The PLA2 activity was eluted in a single peak at 0.4 M-NaCl from a Mono Q anion-exchange column, and migrated with an approximate molecular mass of 70 kDa on a Superose 12 gel-filtration column. The EGF-activated PLA2 activity co-migrated with the basal PLA2 activity upon gel filtration, and persisted after partial purification, which indicates that the activation is due to a stable modification of the enzyme. The EGF-stimulated PLA2 is Ca(2+)-dependent, with maximal activity at micromolar concentrations of Ca2+, has a pH optimum at 9, associates with the particulate cell fraction in a Ca(2+)-dependent fashion, and is selective for arachidonoyl at the sn-2 position. These data demonstrate the EGF-induced activation of a PLA2, which is similar to a recently cloned high-molecular-mass AA-selective cytosolic PLA2, thus providing a link between EGF-receptor tyrosine kinase activation and AA metabolism.


Identification of a dopamine-binding protein on the membrane of the human platelet.

The binding of [3H]dopamine to platelet membranes has been examined in an attempt to identify the putative dopamine-uptake mechanism of the platelet. [3H]Dopamine has been shown to bind to a 42,000 Da glycoprotein in platelet membrane with high affinity (Kd = 22.6 nM) and binding of [3H]dopamine was competed for by dopamine, molecules with catechol moieties, 5-hydroxytryptamine, GSH and ascorbic acid. Differences in pharmacological profile and molecular mass suggest that [3H]dopamine does not bind to a known receptor, a neuronal-type dopamine transporter or the platelet 5-hydroxytryptamine-uptake site. It is proposed that this novel binding site for dopamine, which has been purified 1000-fold from particulate platelet membrane, is likely to be a component of the dopamine-uptake mechanism of the human platelet.