Langley R. Purves

Iron binding proteins of iron-absorbing rat intestinal mucosa.

The distribution and quantitation of the iron-binding proteins of rat small intestinal mucosa was studied, in iron-deficient and replete animals, to explore their role in the absorption of iron. Adsorption (mucosal uptake) of iron in in situ ligated loops of small intestinal mucosa was found to be uniform throughout the length of the small intestine whereas absorption (carcass uptake) showed a steep decreasing gradient from the duodenum to the ileum. The disrupted, in vivo labeled mucosal cells were fractionated by isopycnic centrifugation and transferrin and ferritin were quantitated by radioimmunoassay. Transferrin derived from mucosal cells was shown to have a higher affinity for the antibody than transferrin in serum. Of the transferrin present in the mucosal extract, only a portion could be accounted for by contamination from the serum; the proteolysis resistant and intrinsic transferrin may be mucosal cell specific. Transferrin was found in similar amounts in all regions of the small intestine, was not affected by iron loading but doubled in response to iron deficiency. Mucosal ferritin was found in greater amounts in the iron-absorbing areas of the intestine, increased in the duodenum of iron-loaded animals, and decreased in iron-deficient animals. The incorporation of newly absorbed radioiron into ferritin was only found in iron absorbing regions and was completely inhibited by colchicine and cytochalasin-B, suggesting that ferritin was loaded with iron at the point of iron absorption and that the process is associated with vesicle movement and not simple diffusion. Transferrin and ferritin-specific immunoabsorption and also gel filtration established that no other soluble iron binding proteins were involved in absorption.

Stabilization of the plasmin digestion products of fibrinogen and fibrin by calcium ions

Abstract The role of calcium in the stabilization of fibrinogen and fibrin plasmin digestion products, D-monomer and D-dimer has been studied. The largest D fragment, D1 ∗ , and D-dimer are both stable to further plasmin degradation in the presence of calcium ions. Chelation of calcium leads to further plasmin digestion due to sequential cleavage of peptides from the C-terminal end of the γ-chain. This stabilization is due to calcium ions and not to occupation of the cross-link site. All the digestion products of D-dimer were also found to be stabilized by the re-addition of calcium ions.

Octamer reconstitution from acid-extracted chicken erythrocyte histones

Histone octamers have been reconstituted from acid‐extracted chicken erythrocyte histones. By the criteria of molecular size on exclusion chromatography as well as sedimentation velocity and conformational properties established by circular dichroism, fluorescence spectroscopy and imido‐ester cross‐linking, the reconstituted octamers have a structure identical to that of salt‐extracted octamers.

Properties of the transferrin associated with rat intestinal mucosa.

The transferrin that is isolated from washed intestinal mucosal cell preparations consists partly of a fraction that has properties distinguishing it from serum transferrin. The serum transferrin contaminating mucosal preparations, even when fully saturated with iron and in the presence of proteinase inhibitors, also acquires the properties of the mucosal transferrin when the mucosa is homogenised. The mucosal transferrin is modified by a single cleavage of the polypeptide chain yielding a disulphide-linked peptide of 6550 daltons linked to the parent protein by a disulphide bridge. The amino-terminal sequence of the first 11 residues of this peptide could be aligned with both the known rat and human transferrin carboxy-terminal sequences. In both cases the sequence is preceded by a phenylalanine residue (residue 622 of human transferrin). This suggested that a mucosal chymotryptic enzyme was responsible even though rat transferrin is not susceptible to alpha-chymotrypsin if fully iron-saturated. Since transferrin mRNA is not found in the intestinal mucosa it must be imported from the serum. It remains uncertain whether the modified transferrin is present naturally and plays a role in iron absorption but these findings do indicate the eventual fate of any transferrin imported into an intestinal cell.

The effects of cytoskeletal inhibitors on intestinal iron absorption in the rat

An established and validated method using loops of intestine in vivo in rats was used to study the effects of cytoskeletal inhibitors on iron absorption. Radioactive iron instilled into the loop of intestine pretreated with test substance was monitored in the blood and, after death, ferritin loading with radioactive iron was measured on density gradients of mucosal cell homogenates and absorbed iron in the carcass was determined. Colchicine, vincristine and cytochalasin B all caused dose- and time-dependent inhibition of iron absorption, and the effects of cytochalasin B were reversible within 1 h. It is not known which cellular component is the vehicle for the transcellular movement of iron from the intestinal lumen onto plasma transferrin; however, this study showed that the uptake of iron by ferritin in an iron-absorbing loop of intestine paralleled the actual absorption of iron into the carcass. This phenomenon did not occur in non-iron-absorbing intestinal and was inhibited by the action of the cytoskeletal inhibitors in the iron-absorbing region. Previously we had shown that iron uptake into cells and onto cellular transferrin was virtually the same throughout the small intestine, irrespective of the iron-absorbing capacity of the region. The results of this study therefore suggest that iron absorption depends on an intact cytoskeletal system and that ferritin in the iron-absorbing cell is able to load from the pool of iron committed to transcellular movement onto plasma transferrin.

Acetylcholinesterase of human intestinal tissue affected by Hirschsprung's disease: effect of magnesium chloride on isoforms.

Acetylcholinesterase (AChE) molecular isoforms from anglionic and adjacent unaffected (control) colonic tissue in patients with Hirschsprungs disease (HD) were analysed by density gradient centrifugation in order to determine the major AChE isoforms and the effect of a reported MgCl2 inactivation assay method upon them, with a view to improving the AChE assay used in the diagnosis of Hirschsprungs disease. The total AChE level was greater in the HD-affected colonic tissue than the control tissue (HD: 9.0 vs. control: 7.3 units/g tissue) and this was due to a consistently greater elevation of the globular tetrameric form, G4 (HD: 48.8% vs. control: 35.5% of total AChE). The inactivation of whole tissue homogenates by brief exposure to 4 mol/l MgCl2 followed the same pattern (HD: 48.4% vs. control: 28.7% inactivation). The detergent-extractable G4 is inhibited to a greater extent than the low salt-soluble G4 by exposure to 4 mol/l MgCl2 (83.8% vs. 51.1%). These results imply that the elevated AChE levels in HD are mainly due to increases of the hydrophobic globular tetrameric form of AChE of the same type that is found in differentiating nervous tissue before synapse formation. The monomeric globular isoform G1 is not inhibited but the asymmetric A4, A8 and A12 isoforms are completely inhibited by exposure to 4 mol/l MgCl2. All isoforms of Torpedo (electric ray) electroplax and human erythrocyte AChE, mainly amphiphilic G2, are almost completely inhibited. The inhibition by 4 mol/l MgCl2 of the main G4 isoform in HD-affected intestine accentuates the difference between aganglionic and unaffected intestine in fractionated samples, but does not provide a sufficiently specific G4 isoform assay. The use of 2-4 mol/l MgCl2 in histochemical AChE staining reduces the activity slightly but does not differentiate the tetrameric AChE isoform that is increased in Hirschsprungs disease but does reduce contaminating erythrocyte AChE levels that can obscure the result in blood-stained biopsies. A specific immunochemical stain for hydrophobic AChE tetramers or the associated 20 kDa membrane associated subunit is therefore needed to provide specificity.

The reconstitution of histone H3H4 tetramer from acid extracted histones in the absence of urea

Simple mixing of acid purified histones H3 and H4 in equimolar quantities at low ionic strength near pH 7 does not yield the tetramer but rather a high M r aggregate. Dialysis of acid extracted total or core histones into 2 M NaCl 150 mM phosphate (pH 7.4) followed by fractionation of the histone complexes at lower ionic strength (150 mM NaCl) results in an H3H4 tetramer of a structure identical to that derived from salt‐extracted histones. Dialysis of acid extracted total or core histones directly into the lower ionic strength buffer with subsequent fractionation, results in H3H4 tetramer of closely similar structure.

An abnormal macro lactate dehydrogenase isoenzyme not due to immunoglobulin binding

We report here on the lactate dehydrogenase (LD) of a patient who presented with an additional LD isoenzyme band between LD 3 and LD4 found during routine examination. The abnormal LD was found to be of increased molecular size, which was not due to complexing with immunoglobulins. The abnormal LD was purified by affinity chromatography, labelled and electrophoresed on a SDS-polyacrylamide gel. Autoradiography suggested abnormal aggregation as the cause of increased molecular size.

The effect of Ca2+ on the stability of chicken erythrocyte histone octamers

Histones can be extracted from chicken erythrocyte chromatin with 2 M CaCl2 10 mM Tris (pH 7.4). The core histones so extracted exist as H3–H4 tetramers and H2A–H2B dimers since calcium at >0.5 M result in dissociation of the histone octamer. Reconstitution of octamers occurs on removal of the Ca2+ by dialysis. Although <0.5 M calcium do not result in octamer dissociation, perturbations in the structure can be detected by CD and tyrosine fluorescence spectroscopy.

The carbohydrate prosthetic groups of rat fibrinogen plasmic fragment E.

Rat fibrinogen plasmic fragment E was found to contain one oligosaccharide chain per gamma-chain attached by a glycosylamine linkage. The oligosaccharide was composed of 1 sialic acid, 1 galactose, 2 mannose and 2 glucosamine residues. The probable sequence from the nonreducing end was sialic acid leads to galactose beta leads to mannose alpha leads to mannose alpha leads to glucosamine leads to glucosamine. No difference in the rate of clearance from the rat circulation could be detected between native and desialated fragment E. A non-denaturing method for the purification of fragment E is described.