Insulin-like growth factor I rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells

The type I insulin-like growth factor (IGF) receptor, like the insulin receptor, contains a ligand-stimulated protein-tyrosine kinase activity in its beta-subunit. However, in vivo, no substrates have been identified. We used anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins which occur during IGF-I stimulation of normal rat kidney and Madin-Darby canine kidney cells labeled with ortho[32P]phosphate. Both cells provide a good system to study the function of the type I IGF receptors because they contain high concentrations of these receptors but no insulin receptors. In addition, physiological levels of IGF-I, but not insulin, stimulated DNA synthesis in growth-arrested cells. IGF-I stimulated within 1 min of tyrosine phosphorylation of two proteins. One of them, with a molecular mass between 97 and 102 kDa, was supposed to be the beta-subunit of the type I IGF receptor previously identified. The other protein had an approximate molecular mass of 185 kDa, which resembled, by several criteria, pp 185, originally identified during the initial response of Fao cells to insulin binding (White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186). These data suggest that tyrosine phosphorylation of pp 185 may occur during activation of both the type I IGF receptor and the insulin receptor, and it could be a common substrate that transmits important metabolic signals during ligand binding.     

The major lung surfactant protein, SP 28-36, is a calcium-dependent, carbohydrate-binding protein

SP 28-36, a major protein of pulmonary surfactant, has striking amino acid sequence homology with soluble mannose-binding proteins isolated from rat liver and contains residues common to the carbohydrate-binding domains of other mammalian lectins. We have used carbohydrate-affinity chromatography to investigate carbohydrate-binding properties of SP 28-36 isolated from canine and human (alveolar proteinosis patients) lung lavage. SP 28-36 binds to immobilized D-mannose, L-fucose, D-galactose, and D-glucose. The protein binds only weakly to N-acetyl-D-galactosamine and N acetyl-D-glucosamine. Binding is Ca2+-dependent. The threshold Ca2+ concentration is 0.6 mM and maximal binding occurs with 1 mM Ca2+. Bound protein is quantitatively recovered by elution with 2 mM EDTA. Ba2+, Sr2+, and Mn2+, but not Mg2+, can substitute for Ca2+. Unlike some other mammalian lectins, SP 28-36 binds to carbohydrate at pH 5.0. Recombinant human SP 28-36 isolated from the media of Chinese hamster ovary cells, transfected with a DNA construct encoding SP 28-36, has similar carbohydrate-binding activity to the native proteins. Mannose affinity chromatography of the culture medium of Chinese hamster ovary cells results in an efficient purification of the secreted recombinant human SP 28-36.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

Characterization of an endogenous substrate of the insulin receptor in cultured cells

Using antiphosphotyrosine antibodies, we have characterized the tyrosine phosphorylation of an endogenous substrate of the insulin receptor in Fao hepatoma cells and in Chinese hamster ovary cells transfected with a eukaryotic expression vector containing the human insulin receptor cDNA. In Fao cells, besides the beta-subunit of the insulin receptor, a protein with a molecular mass between 170 and 210 kDa designated pp185, undergoes tyrosine phosphorylation immediately after insulin stimulation reaching a maximum level within 30 s. After 4 h of continuous insulin stimulation, the labeling of pp185 decreased to less than half of its original intensity, whereas the insulin receptor was unchanged. After 24 h of insulin stimulation, the phosphotyrosine-containing insulin receptor decreased by 75% owing to down-regulation, whereas the pp185 was completely undetectable. By several biochemical and physiological criteria, the pp185 is distinct from the insulin receptor. The pp185 and the beta-subunit of the insulin receptor were strongly labeled with [32P]orthophosphate, but in contrast to the insulin receptor, the pp185 was not labeled by cross-linking with 125I-insulin or surface 125I iodination. Unlike the insulin receptor, the pp185 was extracted from Fao cells without detergent, and tryptic phosphopeptide mapping of the pp185 and the insulin receptor yielded distinct patterns. Thus, the pp185 is not located at the external face of the plasma membrane and does not bind insulin. Treatment of Fao cells with the phorbol ester, phorbol 12-myristate 13-acetate, stimulated the phosphorylation of two proteins with molecular weights of 170 and 210 kDa which were immunoprecipitated with the anti-phosphotyrosine antibody. Subsequent insulin stimulation increased the phosphorylation of the 210 kDa protein, but the pp185 was not detected. Increasing the concentration of the human insulin receptor in the Chinese hamster ovary cells by transfection with a plasmid containing the human insulin receptor cDNA caused a higher level of tyrosine phosphorylation of the beta-subunit and the pp185. These data support the notion that the insulin signal may be transmitted to a cellular substrate (pp185) which may initiate insulin action at intracellular sites.     

Spasmodic, a mutation on chromosome 11 in the mouse

A new recessive mutation, spasmodic (spd), producing behavior that mimics that of the neurological mutation spastic (spa) with rapid tremors, stiff posture, and difficulty in righting, arose spontaneously in strain A/HeJ at the Jackson Laboratory in 1979. It is not an allele of spa and linkage tests show that this mutation is located close to vestigial tail (vt) near the center of chromosome 11. Additional genetic tests show that it is not an allele of trembler (Tr), shaker-2 (sh-2), nor vibrator (vb), all neurological mutations located in the same region of chromosome 11. No differences were observed in the levels of the major CNS and PNS myelin proteins or lipids of spd/spd mice versus littermate controls, suggesting that, unlike several closely linked mutations, the spd mutation does not affect myelination. Pharmacological studies reported here show that aminooxyacetic acid improves the behavioral abnormalities of affected spd/spd mice in the same way it improves the behavior of affected spa/spa mice. However, unlike the spa/spa mice, there are no changes in the postsynaptic receptors for glycine, GABA, or benzodiazepines in spd/spd mice.     

Hominid footprints at Laetoli: facts and interpretations

The history of discovery and interpretation of primate footprints at the site of Laetoli in northern Tanzania is reviewed. An analysis of the geological context of these tracks is provided. The hominid tracks in Tuff 7 at Site G in the Garusi River Valley demonstrate bipedality at a mid-Pliocene datum. Comparison of these tracks and the Hadar hominid foot fossils by Tuttle has led him to conclude that Australopithecus afarensis did not make the Tanzanian prints and that a more derived form of hominid is therefore indicated at Laetoli. An alternative interpretation has been offered by Stern and Susman who posit a conforming "transitional morphology" in both the Tanzanian prints and the Ethiopian bones. The present examines both hypotheses and shows that neither is likely to be entirely correct. To illustrate this point, a reconstruction of the foot skeleton of a female A. afarensis is undertaken, and the results are compared to the Laetoli tracks. We conclude that A. afarensis represents the best candidate for the maker of the Laetoli hominid trails.     

Phosphorylation heterogeneity of tryptic phosphopeptides of chicken riboflavin-binding protein

The tryptic phosphopeptide of hen egg white riboflavin-binding protein has been found to exist as a mixture of peptides which differ only with respect to the number of covalently bound phosphoryl groups. Anion-exchange chromatography was used to separate homologues of the tryptic phosphopeptide of egg white riboflavin-binding protein. Four peptide peaks were obtained and analyzed using plasma desorption mass spectrometry. Molecular ions obtained agree closely with calculated molecular weight values for phosphopeptides with 8, 7 and 5 phosphoryl groups. Amino acid analyses showed that the octa- and hepta-phosphorylated peptides were pure and had the same amino acid compositions.     

The entrapment of [14C]ascorbic acid in human erythrocytes

Radioactively labelled ascorbic acid and dehydroascorbic acid, when incubated with human blood, migrate irreversibly into human red blood cells. Isolation and characterization of the moieties trapped within the cells via infrared spectroscopy established both their identities as L-ascorbic acid. Evidence in the form of the degree of in vitro entrapment of ascorbic acid as a function of the times of incubation and the effect of incubation temperature, anion recognition site inhibitor, and active transport inhibitor on the rate of entrapment support the hypothesis that ascorbic acid is oxidized on or near the surface of the red blood cell to dehydroascorbic acid which migrates through the lipid portion of the cell wall and is reduced back to ascorbic acid within the cell. The resulting L-ascorbic acid can not pass through the cell wall and is therefore entrapped.