Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility

Axoplasm from the squid giant axon contains a soluble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine brain. The purification of the translocator protein depended primarily on its unusual property of forming a high affinity complex with microtubules in the presence of a nonhydrolyzable ATP analog, adenylyl imidodiphosphate. The protein, once released from microtubules with ATP, migrates on gel filtration columns with an apparent molecular weight of 600 kilodaltons and contains 110-120 and 60-70 kilodalton polypeptides. This protein is distinct in molecular weight and enzymatic behavior from myosin or dynein, which suggests that it belongs to a novel class of force-generating molecules, for which we propose the name kinesin.     

Neuron-schwann cell interaction in basal lamina formation

The availability of tissue culture systems that allow the growth of nerve cells, Schwann cells, and fibroblasts separately or in various combinations now makes possible investigation of the role of cell interactions in the development of the peripheral nervous system. Using these systems it was earlier found that basal lamina is formed on the Schwann cell surface in cultures of sensory ganglion cells and Schwann cells without fibroblasts. It is here reported that the presence of nerve cells is required for the generation of basal lamina on the Schwann cell plasmalemma. Utilizing nerve cell-Schwann cell preparations devoid of fibroblasts, this was found in the following ways. (1) When nerve cells are removed from 3- to 5-week-old cultures, the basal lamina disappears from Schwann cells. (2) If nerve cells are added back to such Schwann cell populations, Schwann cell basal lamina reappears. (3) Removal of nerve cells from older (3–4 months) cultures does not lead to basal lamina loss; areas presumed not to have been coated with lamina before neurite degeneration remain so, suggesting that the lamina persists but is not reformed. (4) If basal lamina is removed with trypsin, it is reformed in neuron plus Schwann cell cultures but not in Schwann cell populations alone. Thus, the formation but not the persistence of Schwann cell basal lamina requires the presence of nerve cells.     

Effects of catecholamines on ammoniagenesis and gluconeogenesis by renal cortex in vitro

Norepinephrine (arterenol) and a synthetic catecholamine, isoproterenol, increase the production of ammonia and glucose from glutamine and glutamate by rat renal cortical slices in vitro. The stimulation of both ammonia and glucose production by isoproterenol was greater than that observed with identical molar concentrations of arterenol. Isoproterenol markedly increased the concentration of cyclic AMP in rat renal cortical slices. Addition of propranolol, a β-adrenergic blocking agent, prevented the increase of cyclic AMP levels induced by isoproterenol. Cyclic AMP increased both ammoniagenesis and gluconeogenesis by kidney cortex. Thehe increase in ammonia production produced by isoprotenol was blocked by the addition of propranolol. It is concluded that the increase in ammonia and glucose production caused by isoproterenol is mediated through the release of cyclic AMP.     

Augmentation of IgE-mediated release of histamine by 5-hydroxyeicosatetraenoic acid and 12-hydroxyeicosatetraenoic acid

Highly purified rat mast cells converted 1.12% and 1.64% of exogenously added [1-¹⁴C]arachidonic acid to 5-OH-6,8,11,14 eicosatetraenoic acid (5-HETE) and 12-OH-5,8,10,14 eicosatetraenoic acid (12-HETE) respectively during a three minute incubation at 37°. Both 5-HETE and 12-HETE (1–10 μM) augmented the histamine release response to goat anti-rat IgE antibody (a reverse anaphylaxis system). These results indicate that mast cells synthesize 5-HETE and 12-HETE and that these molecules can enhance mediator release.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Effect of N,N,N',N'-tetramethylethylenediamine on the migration of proteins in SDS polyacrylamide gels

Changing the concentration of TEMED in SDS polyacrylamide gels was found to affect the migration of proteins. Elevation of TEMED levels caused a generalized decrease in mobility with some proteins being affected more than others. With various brain protein preparations this differential effect could be used to improve the separation of adjacent protein bands. In additon, it was found that a change in the TEMED concentration affected the results of molecular weight determinations. The effect of TEMED was also observed in one non-SDS system.     

Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Effects of cultured astroglia on the survival of neonatal rat retinal ganglion cells in vitro

Retinal ganglion cells (RGC), as identified by retrograde horseradish peroxidase (HRP) labeling technique, were cultured in a minimal medium; only 20% of them survived after 16 hr in vitro. However, superior colliculus-conditioned medium was capable of supporting 100% of RGC over this assay time; enhanced neurite expression also was evident. It was decided to investigate whether glial cells within the superior colliculus may provide a soluble factor capable of supporting RGC. Glial-conditioned medium prepared over monolayers of either predominantly flat astrocytes (relatively immature) or predominantly process-bearing (mature) astrocytes failed to maintain RGC. The possibility that astrocytes may provide support for RGC via membrane contact was then investigated. Dissociated retinae were grown on monolayers consisting primarily of either flat or process-bearing astrocytes. Cultures rich in flat astrocytes maintained over 70% of RGC originally present, and many of them exhibited extensive neurite outgrowth and elongation. Process-bearing astrocytes were unable to support RGC survival. Immature astroglial cells may therefore support RGC via glial-neuronal interaction.     

Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct

When meiotic maturation of primary oocytes of the starfish Asterias forbesi is induced by 1-methyladenine, rapid and striking changes in the pattern of protein synthesis detectable by electrophoresis occur after germinal vesicle breakdown. These include a decline in relative labeling with [³⁵S]methionine of several polypeptides synthesized in the oocyte, and increased labeling and new appearance of several polypeptides. Fertilization does not result in other detectable changes. The population of total mRNA translatable in a rabbit reticulocyte lysate cell-free system does not change, but the distribution of mRNAs between polysomes and the postribosomal supernatant reflects the changes observed in vivo. Thus these changes are regulated at the translational level. A review of the literature indicates that translationally mediated changes in patterns of protein synthesis during maturation of oocytes may be a widespread phenomenon.     

Cascade control of E. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade.

Enzymes and regulatory proteins involved in the cascade control of glutamine synthetase activity of Escherichia coli have been separated from one another and the effects of numerous metabolites on each step in the cascade have been determined. The adenylyl transferase (ATase) -catalyzed adenylylation of glutamine synthetase, which requires the presence of the unmodified form of the regulatory protein PII is enhanced by glutamine and is inhibited by either α-ketoglutarate (α-KG) or the uridylylated form (PII·UMP) of the regulatory protein. PII·UMP and α-KG act synergistically to inhibit this activity. In contrast, the PII·UMP-dependent, ATase-catalyzed deadenylylation of glutamine synthetase requires α-KG and ATP and is inhibited by glutamine or PII and synergistically by glutamine plus PII. The capacity of uridylyl transferase (UTase) to catalyze the uridylylation of PII is dependent on the presence of α-KG and ATP and is inhibited by glutamine. The deuridylylation of PII·UMP by the uridylyl removing enzyme (UR) is enhanced by glutamine but is unaffected by α-KG. However, CMP, UMP, and CoA all inhibit activity at 10^?6m. High concentrations of ATase inhibit both UR and UTase activities, presumably by binding the regulatory protein. Of more than 50 substances that alter the activity of at least one enzyme in the cascade, only α-KG and glutamine affect the activity at every step. This accounts for the observation that glutamine synthetase activity in vivo is very sensitive to the intracellular ratio of α-KG to glutamine.     

A sensitive assay employing a solid-phase metal chelator to estimate rates of iron accumulation by ferritin

To minimize the contribution by the appreciable rates of extraferritin Fe(II) autoxidation/polymerization which proceed concomitant with the accumulation of iron by ferritin, a simple discontinuous, spectrophotometric kinetic assay was developed. This assay procedure utilizes a commercial metal chelator to complex the substrate and thus quench the reaction. Because operation is between 0 and 20°C and below 1.5 mm Fe(II), interference by autoxidation is negligible. Rates are observed to be first order with respect to apoferritin concentration and first order with respect to Fe(II) concentration at substrate-to-protein ratios as great as 2 × 10⁴. Between 0 and 20°C, the temperature dependence of the rate yielded a linear Arrhenius plot with an activation energy of + 15.2 kcal mol^?1. The advantages offered by this assay procedure over existing assays are (i) the assay can be up to an order of magnitude more sensitive, and (ii) the interference by extraferritin Fe(II) autoxidation is insignificant.     

Inhibition of plasma prolactin in the rat by amantadine

A single iv injection of 15 or 30 but not 7.5 mg/kg BW of an antiviral drug, amantadine, significantly (P less than 0.05) decreased plasma prolactin (PRL) concentrations in male rats. This effect was dose-dependent, with the highest dose producing a longer-lasting decrease in plasma PRL. The amantadine-induced decrease was unaffected by a simultaneous injection of 5-hydroxytryptophan (30 mg/kg BW) but was completely blocked by a simultaneous injection of haloperidol (0.05 mg/kg BW). It is concluded that this novel effect of amantadine on PRL is produced by an interaction with the dopaminergic system.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

15-ketoprostaglandin delta13 reductase from human placenta: purification, kinetics, and inhibitor binding.

An NADH-dependent 15-ketoprostaglandin Δ¹³ reductase has been purified to near homogeneity from human placenta by a procedure which includes affinity chromatography on blue Sepharose. The enzyme utilizes as substrates 15-ketoprostaglandins of the E, F, A, and B series, and the reaction is experimentally irreversible. Molecular weight estimations on Sephadex G-100 and sodium dodecyl sulfate disc gel electrophoresis suggest that the enzyme is a dimer. The subunits appear to be similar in size if not identical and have a molecular weight of 35,000. The mechanism of the reaction of 15-ketoprostaglandin E₂ and NADH catalyzed by this enzyme has been investigated by steady-state kinetic methods. The 13,14-dihydro-15-ketoprostaglandin product is an inhibitor of the reaction, being competitive with respect to 15-ketoprostaglandin E₂ and noncompetitive with respect to NADH; NAD⁺ does not inhibit the reaction. NADPH and Cibacron blue 3G-A are “dead-end” inhibitors of the reaction; both act competitively with respect to NADH and noncompetitively with respect to 15-ketoprostaglandin E₂. These observations are consistent with a rapid equilibrium random mechanism with the formation of an unreactive enzyme · NADH · 13,14-dihydro-15-ketoprostaglandin E₂ complex. The interaction of NADPH and Cibacron blue 3G-A with the free enzyme was investigated further by fluorimetry. Both substances bind to the free enzyme and quench its fluorescence. This property was utilized to titrate the enzyme, and a value of 3.28 × 10^?11 mol of binding sites/mU of enzyme was obtained.     

Defective phosphorylation and processing of beta-hexosaminidase by intact cultured fibroblasts from patients with mucolipidosis III

Previous studies of the synthesis, phosphorylation, and processing of β-hexosaminidase in cultured fibroblasts from normal individuals and from patients with mucolipidosis II (I-cell disease) (A. Hasilik and E. F. Neufeld, 1980, J. Biol. Chem.225, 4937–4946) have been extended to fibroblasts derived from patients with a related genetic disorder, mucolipidosis III (pseudo-Hurler polydystrophy). The enzyme was biosynthetically labeled in pulse-chase experiments with [³H]leucine and ³³P_i, and isolated from cells and medium by immunoprecipitation. The constitutent α and β chains of the enzyme were separated by polyacrylamide gel electrophoresis under reducing and denaturing conditions, visualized by autoradiography and fluorography, extracted from the gel, and quantitated by liquid scintillation spectrometry. Enzyme produced by fibroblasts from mucolipidosis III patients had a very low but detectable phosphate content; a high proportion of newly made enzyme was secreted, though some remained within the cells and was processed to mature enzyme; the presence of NH₄Cl during the labeling and chase did not significantly increase the amount of enzyme secreted. The β-hexosaminidase produced by mucolipidosis III fibroblasts thus resembled more closely that produced by fibroblasts from patients with mucolipidosis II than the normal enzyme. β-Hexosaminidase made by fibroblasts from mucolipidosis II heterozygotes was similar to the normal enzyme with respect to phosphorylation, processing, and secretion. Mucolipidosis II and III fibroblasts could endocytose normal precursor β-hexosaminidase and process it to the mature form. The deficiency of mature enzyme in the patients' cells may therefore be attributed to failure of the unphosphorylated enzyme to be incorporated into lysosomes, where processing would normally occur.