An intramembranous reductant which participates in the proline hydroxylation reaction with intracisternal prolyl hydroxylase and unhydroxylated procollagen in isolated microsomes from L-929 cells

We previously have described a substance present in crude sonicates of L-929 cells which replaced ascorbate in vitro as a reductant for prolyl hydroxylase (B. Peterkofsky, D. Kalwinksy and R. Assad, 1980, Arch. Biochem. Biophys.199, 362–373). In the present study we found that almost 90% of the substance was particulate after differential centrifugation of stationary phase L-929 cell homogenates. The substance was not localized in nuclei or mitochondria and was found in the same fractions as microsomes, but these fractions also contained lysosomes and cell membranes. The reductant could not be solubilized from particles by Brij-35, indicating that it is an intrinsic component of a membrane rather than intracisternally located. The intramembranous cofactor, in the absence of ascorbate, participated in the in vitro hydroxylation of [4-³H]proline in radio-actively labeled, intracisternal unhydroxylated procollagen in isolated microsomes which also contained prolyl hydroxylase. Hydroxylation was determined by measuring tritiated water formed from release of the 4-trans tritium atom. Since it is unlikely that such participation could occur if the cofactor were located within the membrane of another subcellular organelle, we have concluded that it is in the same particle as prolyl hydroxylase and unhydroxylated procollagen, that is, the microsome. With the endogenous reductant the reaction was slower than with saturating ascorbate and was increased by NADH. Maximum hydroxylation with the endogenous reductant was close to that which could be achieved with ascorbate. These results provide strong evidence that the endogenous reductant alone can account for the phenomenon of ascorbate-independent proline hydroxylation in L-929 cells. As in the case of ascorbate, the microsomal reductant functioned only in the presence of α-ketoglutarate and Fe²⁺ and served as reductant for lysyl hydroxylase. It also was detected in the particulate fraction of virally transformed BALB 3T3 cells and in purified microsomes from bones of intact chick embryos. Since ascorbate could be taken up and concentrated in bone microsomes, it is unlikely that the endogenous reductant serves as an intermediary between ascorbate and intracisternal prolyl hydroxylase.     

The alpha-glucosidases of Dictyostelium discoideum. II. Developmental regulation and cellular localization

Four isozymes of α-glucosidase in Dictyostelium discoideum have been identified and some of their enzymatic and physical properties characterized (R. H. Borts and R. L. Dimond, 1981, Develop. Biol.87, 176–184). In this report the cellular localization and developmental regulation of three of these isozymes are determined. α-Glucosidase-1 is the major isozyme of vegetative amoebae. It is lysosomally localized and secreted from the cell under certain conditions. It has an acidic pH optimum and carries the common antigenic determinant found on all lysosomal enzymes in this organism. The specific activity of this isozyme begins to decrease within a few hours after the initiation of development and is no longer detectable in the mature fruiting body. α-Glucosidase-2 has a neutral pH optimum and is neither lysosomal nor secreted. Rather it is membrane bound and is possibly located on the cisternal side of microsomal vesicles. This isozyme does not possess the common antigenic determinant. α-Glucosidase-2 comprises 20–40% of the total α-glucosidase activity of the vegetative cell. Its specific activity increases threefold during development. This isozyme appears to be developmentally controlled since it fails to accumulate in aggregation deficient mutants. Its accumulation is also dependent upon continued protein synthesis. α-Glucosidase-4, like α-glucosidase-1, has an acidic pH optimum. It does not appear to be lysosomally localized nor membrane bound. Approximately 30% of the activity is precipitable by antibody against the common antigenic determinant indicating that it is less highly modified or fewer molecules are modified. The isozyme is undetectable during vegetative growth and does not begin to accumulate until late aggregation. Activity peaks in mature fruiting bodies where it is the predominant acidic α-glucosidase activity. Accumulation of α-glucosidase-4 is blocked in morphologically deficient mutants and by inhibitors of protein synthesis.     

Detection of inactive or less active forms of tyrosine hydroxylase in human adrenals by a sandwich enzyme immunoassay

A sensitive sandwich enzyme immunoassay for tyrosine hydroxylase (TH) from bovine and human adrenals has been developed. Anti-TH antibody was prepared from bovine adrenal TH. The assay system consisted of an antibody F(ab')2 immobilized on polystyrene beads as a solid phase and of beta-D-galactosidase-conjugated antibody. This method was highly sensitive and specific for the assay of TH. Human adrenal TH level was determined by similar sensitivity as bovine adrenal TH, suggesting the presence of common antigenic sites between human and bovine adrenal enzymes. The presence of inactive or less active forms of TH in human adrenals was revealed by purification of the enzyme and monitoring with this enzyme immunoassay as well as with enzyme activity assay.     

Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts

Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.     

A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

A spontaneously active (Mr greater than 350,000) and an ATPMg-dependent phosphatase (Mr congruent to 140,000) were identified in bovine aortic smooth muscle. The spontaneously active phosphatase was effective in dephosphorylating both phosphorylase a (240nmol32P/min/mg) and phosphorylated myosin light chains (1000nmol32P/min/mg). In contrast, the ATPMg-dependent phosphatase was only effective in dephosphorylating phosphorylase a (400nmol32P/min/mg). Phosphorylase phosphatase activity of the ATPMg-dependent enzyme was suppressed by the well-characterized modulator protein (inhibitor-2), whereas the activity of the spontaneously active enzyme was unaffected. The aortic spontaneously active phosphatase did not convert to an ATPMg-dependent form when it was stored at 4 degrees or incubated at 30 degrees C in either the presence or absence of modulator protein. These findings suggest that spontaneous and ATPMg-dependent phosphatase activities described in these studies are probably ascribable to different enzymes. Since both phosphorylase and myosin light chains are phosphorylated when smooth muscle contracts these phosphatases may participate in coordinating arterial contractility and metabolism.     

Simultaneous simple purification of tyrosine hydroxylase and dihydropteridine reductase

A rapid and simple simultaneous micropurification procedure of tyrosine hydroxylase (TH) and dihydropteridine reductase (DPR) was developed from soluble supernatants of 1 to 2 g of rat adrenal gland or caudate nucleus. All purification procedures for the two enzymes were complete within 3 days. The recovery of TH and DPR was reproducible and approximately 20 and 40%, respectively. Purification procedure for TH involved chromatographies with DEAE-Sephacel, Bio-Gel A-1.5 m, and heparin-Sepharose. As judged by gel filtration and sodium dodecyl sulfate-gel electrophoresis, the enzyme purified from each tissue appeared to be homogeneous and was composed of an identical subunit, each possessing a Mr of 60,000. With DEAE-Sephacel column chromatography, TH was separated completely from DPR. DPR was purified by subsequent chromatographies with Sephadex G-50 and blue Sepharose to a purity of 50%. DPR in adrenals and brain was found to be a NADH-dependent type. This micropurification procedure is applicable to assessing the molecular properties of TH modified physiologically or pharmacologically in vivo, and to getting a small amount of the pure enzyme as antigen for producing its antibody.     

Mechanism of tyrosine hydroxylase activation by phosphorylation

It was found that the fluorescence of 1,N⁶-ethenoadenosine triphosphate (ε-ATP) bound to myosin subfragment-1 (S-1) is resistant to quenching by acrylamide, while free ε-ATP is effectively quenched. Thus in the presence of acrylamide the bound ε-ATP is still highly fluorescent, while free ε-ATP is much less fluorescent. The Stern-Volmer constants of bound and free ε-ATP are 6.83 and 57.86 M^?1, respectively. Therefore it is easy to distinguish spectro-scopically the nucleotide-ligated S-1 from nucleotide-free S-1. Moreover acrylamide does not alter the S-1-Mg²⁺-ε-ATPase behavior.     

The subcellular localization of calmodulin, cyclic AMP phosphodiesterase, and adenylate cyclase in bovine adrenal medulla

The subcellular localization of calmodulin, cyclic nucleotide phosphodiesterase, and adenylate cyclase was studied in bovine adrenal medulla. Approximately 70% of the calmodulin and 90% of the cAMP phosphodiesterase activities were found colocalized in the cytoplasm. The subcellular distribution of adenylate cyclase closely paralleled the distribution of acetylcholinesterase, a marker for plasma membranes. The fraction of calmodulin which is particulate in nature has a distribution profile very similar to that of adenylate cyclase. The chromaffin granule fraction contained only 0.86% of the total cAMP phosphodiesterase, 0.41% of the total adenylate cyclase, and 1.4% of the total calmodulin.     

Multiple sites for the initiation of microtubule assembly in mammalian cells.

The pattern of microtubule regrowth in mammalian fibroblast and epithelial cells has been examined by immunofluorescence of cytoskeletal preparations with antibody to tubulin. After reversal of treatment with colcemid, vinblastine or low temperature, microtubules appear to grow simultaneously from several distinct initiation sites located within 5 microns of the nucleus of mouse and human fibroblasts. Each site initiates the growth of 10-30 microtubules. More than 70% of the mouse fibroblasts have between 5 and 10 initiation sites with an average of 8. The human fibroblasts have an average of 5 sites per cell. The average number and numerical distribution of sites per fibroblast cell are not affected by time of exposure to colcemid or the concentration of colcemid applied to the cells. Multiple microtubule initiation sites are also observed during the process of microtubule depolymerization. In addition to growth from these complex initiation sites, microtubules appear to grow singly from the perinuclear region of human fibroblasts. The regrowth of individual microtubules from the perinuclear growth is especially prominent in epithelial cell lines from rat kangaroo and pig. These epithelial lines have only a single complex initiation site per cell. Two classes of complex initiation sites can be distinguished in microtubule regrowth experiments in human and mouse fibroblasts after exposure to griseofulvin. Microtubules first grow extensively from a single distinct site, which has approximately 20 microtubules growing from it and may be the centriole or centriolar pair. Subsequently, microtubules regrow from other perinuclear complex initiation sites. It thus appears that at least three distinct classes of initiation sites can be observed in mammalian cells: primary sites, which regrow microtubules first after griseofulvin treatment; secondary sites, which are distinct perinuclear sites and recover from griseofulvin treatment more slowly than the primary sites; and tertiary sites or sites of growth of single microtubules, also located near the cell nucleus.     

Purification and properties of purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.     

An extracellular retinol-binding glycoprotein in the rat eye—characterization,localization and biosynthesis

We have identified and partially purified interstitial retinol-binding protein (IRBP) from the subretinal space of the rat. It appeared to be glycosylated. Its apparent mol. wt was 270,000 by gel-filtration and 144,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rat IRBP cross-reacted with anti-bovine IRBP sheep and rabbit sera, bound all-trans-[15-³H] retinol and was bound by concanavalin A. IRBP was not detected in the cytosols of the neural retina or retinal pigment epithelium and choroid. This distribution was confirmed by immunocytochemistry using a fluorescence-labeled second antibody. Immunospecific fluorescence was most intense in the interphotoreceptor matrix in a 6.5 μm band adjacent to the retinal pigment epithelium. It was less intense over the remainder of the rod outer segment layer and was comparatively faint over the inner segment region. Its occurrence in the interstitial space between the photoreceptors and retinal pigment epithelium coupled with the fact it bound all-trans-[15-³H] retinol supports the concept that it may be implicated in the transport of retinoids between the retina and the retinal pigment epithelium during the visual cycle. When incubated with [³H]leucine or [³H]glucosamine, isolated retinas (but not retinal pigment epithelium and choroid) secreted labeled IRBP into the medium. This suggests that the retina plays a role in regulating the amount of IRBP in the subretinal space.     

The genome-linked protein of picornaviruses. VII. Genetic mapping of poliovirus VPg by protein and RNA sequence studies

The poliovirus genome-linked protein (VPg) has been subjected to radiochemical microsequence analysis. Sequence studies of virion RNA by a modification of Sanger's dideoxy method have revealed a base sequence corresponding to the amino acid analysis. This result proves that VPg is virus-encoded. The RNA sequence has allowed us to predict the total amino acid sequence of VPg and part of its precursor. VPg is, at most, 27 amino acids long. It maps within the 3' terminal segment of the viral genome that encodes the precursor polypeptide NCVP1b for the virus-specific RNA polymerase NCVP4.     

Studies on the recognition of xenogeneic cells by nonimmune macrophages. I. Destruction of chicken fibroblasts in vitro by murine macrophages.

Human cord blood lymphocytes were compared with adult lymphocytes with regard to proportions of cells with surface markers for surface immunoglobulin (Ig), receptors for C′3 and the Fc-portion of IgG, as well as two types of erythrocyte rosettes (rapid and late E-rosettes). A significant decrease (P < 0.02 ? 0.05) in both early and late E-rosettes was noted when cord cells were compared to adult lymphocytes. After 20 hr of incubation at 37 °C, proportions of cells bearing Fc receptors in cord blood samples showed striking increments (P < 0.001) when compared with adult lymphocytes. T cell enrichment studies and sequential depletion of cells bearing Fc receptors as well as E-rosette forming cells indicated that the precursors of cells generating Fc receptors in vitro did not arise from cells with Fc receptors or T cell markers.     

The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture

The production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl-L-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 +/- 1.4% to 10.5 +/- 2.4% with 10 microM TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 microM TLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 micrograms/ml leupeptin or 60 micrograms/ml pepstatin A. However, the production of collagen was reduced to 10.9 +/- 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 microM TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.     

Tissue fractionation in rat brain, kidney and liver. I. Intracellular localization of a 5-methyltetrahydrofolic acid requiring enzyme.

The intracellular distribution of N-methyl-transferase requiring 5-methyl-tetrahydrofolic acid (5 MT-NMT) was studied in brain, kidney and liver of rats. Among these different tissues, the kidney displayed the highest enzyme activity, more than 20 times the activity detected in the brain. As the striatum and, to a lesser extent the hypothalamus, were found to contain slightly higher 5 MT-NMT than other cerebral regions, they were also selected for the study of the subcellular localization. Tissue fractionation was performed by differential centrifugation yielding five different fractions which were analyzed for their enzymatic content not only of 5 MT-NMT but also of marker enzymes, such as cytochrome oxidase, acid phosphatase and inosine diphosphatase. In all the tissues studied, 5 MT-NMT was recovered in the supernatant fraction. Therefore one may consider this enzyme to belong to the cytosol. Although a neuronal localization cannot be excluded, it is beyond doubt that the enzyme is contained in other cellular types. In the brain fractionation, the five fraction procedure seems to be very useful especially when the subcellular distribution of a given enzyme is compared to that obtained in other tissues like liver or kidney. Finally 5 MT-NMT may be considered a good marker enzyme for the supernatant fraction.     

Control of cellular redox potential as measured in a steady-state, cell-free system

A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.     

Properties and subcellular localization of adenosine diphosphatase in rat heart

Some properties and subcellular localization of adenosine diphosphatase (ADPase) activity from rat heart have been investigated. The pH optimum was 7.4, maximal activity was found with 5 mM MgCl2, and the apparent Km was 20 microM. ADPase activity was strongly inhibited by NaF and AppNHp, and to a lesser extent by AMP and GppNHp. The enzyme was not inhibited by p-nitrophenylphosphate, beta-glycerophosphate, or pyridoxal phosphate. The distribution of ADPase activity in subcellular fractions obtained by differential centrifugation parallel ouabain-sensitive (Na+-K+)ATPase and 5'-nucleotidase activities, suggesting a plasma membrane-bound localization. The functional significance of ADPase in adenosine production and hemostasis is discussed.