Native-like intermediate on the folding pathway of Escherichia coli succinyl-CoA synthetase

The transition between the native and denatured states of the tetrameric succinyl-CoA synthetase from Escherichia coli has been investigated by circular dichroism, fluorescence spectroscopy, cross-linking by glutaraldehyde and activity measurements. At pH 7.4 and 25 degrees C, both denaturation of succinyl-CoA synthetase by guanidine hydrochloride and refolding of the denatured enzyme have been characterized as reversible reactions. In the presence of its substrate ATP, the denatured enzyme could be successfully reconstituted into the active enzyme with a yield of 71-100%. Kinetically, reacquisition of secondary structure by the denatured enzyme was rapid and occurred within 1 min after refolding was initiated. On the other hand, its reactivation was a slow process which continued up to 25 min before 90% of the native activity could be restored. Both secondary and quaternary structures of the enzyme, reconstituted in the absence of ATP, were indistinguishable from those of the native enzyme but the renatured protein was catalytically inactive. This observation indicates the presence of catalytically inactive tetramer as an intermediate in the reconstitution process. The reconstituted protein could be reactivated by ATP even 10 min after the reacquisition of the native secondary structure by the refolding protein. However, reactivation of the protein by ATP 60 min after the regain of secondary structure was significantly less, suggesting that rapid refolding and reassociation of the monomers into a native-like tetramer and reactivation of the tetramer are sequential events; the latter involving slow and small conformational rearrangements in the refolded enzyme that are likely to be associated with phosphorylation.     

Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin

Receptor-mediated endocytosis of 125I-insulin and 125I-prolactin into liver parenchymal cells has been studied by quantitative subcellular fractionation. Differential centrifugation yielded three particulate fractions, N (nuclear), ML (large granule), and P (microsomes), and a final supernatant (S). Quantitative differences in the extent and rates of accumulation of 125I-insulin and 125I-prolactin into the fractions were observed. The acidotropic agent chloroquine and the microtubule disrupting agent colchicine were administered separately to rats. The agents increased significantly the T 1/2 of hormone clearance from the liver and augmented the accumulation of both ligands in the low-speed ML fraction. However, differences in the rates of accumulation of insulin and prolactin into all cell fractions were still maintained. Analytical centrifugation of each of the particulate fractions was carried out in order to determine if different endocytic components were specific to insulin or prolactin internalization. This was not the case. An "early" endosomal component of density 1.11 was identified in microsomes. A "late" endosome of density 1.10 was identified in the large granule (ML) fraction. Both endosomal components appeared to accumulate insulin and prolactin but at different rates. Marker enzyme analysis identified the presumed plasma membrane component in microsomes (density approximately 1.155). This component showed a significant difference in the rate of loss of 125I-insulin (T 1/2 approximately 4.1 min) as compared to that of 125I-prolactin (T 1/2 approximately 12.7 min). A further difference in the handling of the ligands was observed in early endosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential kinetics and sensitivity to chloroquine of receptor-mediated insulin and prolactin endocytosis in liver parenchymal cells

Systemically injected [125I]prolactin or [125I]insulin was accumulated and cleared from rat liver at different rates. Quantitative subcellular fractionation indicated a predominant accumulation of [125I]insulin in liver microsomes while [125I]prolactin was found in both the light-mitochondrial and microsomal fractions. The acidotropic agent chloroquine diminished the rate and extent of loss of each ligand from liver homogenates. In chloroquine treated rats, radiolabeled insulin accumulated in both the light-mitochondrial and the microsomal fractions. Subfraction of microsomes on discontinuous sucrose gradients revealed "early' endosomes in which ligand uptake was maximal at 2-5 min. In contrast, comparable subfraction of the of light mitochondrial fraction revealed "late' endosomes in which ligand uptake was maximal at 10-20 min. Chloroquine-treated rats showed a more marked enhancement of insulin compared to prolactin uptake in the "early' endosomes. It is suggested that "early' endosomes found in the Golgi-intermediate and -heavy fractions floated from parent microsomes may selectively degrade insulin but not prolactin. This could account for the apparently different kinetics of insulin and prolactin uptake into liver parenchyma.     

Hydrobiology and organic production in a marl lake of Kashmir Himalayan Valley

L. Naranbagh (alt. 1587 m) is a polymictic, shallow marl lake in the flood-plain valley of Kashmir, India. Macrofloral affinities resemble Potamogeton Type of Forsberg (1965) with alkaline waters, not rich in phosphorus. CaCO₃ precipitation coupled with decline in Ca²⁺ and alkalinity values are characteristic of the lake. Fluctuations in Mg²⁺, Na⁺, K⁺, and Cl^– were relatively conservative. The levels of PO _inf4 ^sup3– -P and NO _inf3 ^sup– -N indicate moderate fertility of the lake water.Persistence of a summer-autumn planktonic algal pulse is related to favourable irradiance, high water temperatures, and increased photosynthetic efficiency values. The most striking seasonality in photosynthetic rates (m^–2 h^–1) between winter minimum (3 mg C_assim) and summer maximum (75.4 mg C_assim) is determined by mainly climatic changes. Energy flow gave annual phytoplankton production of 51.95 × 10² KJ m^–2 for the ecosystem.The nutrient levels and productivity rates suggest mesotrophic status of L. Naranbagh in classic oligoeutrophic classification of lake types.     

Cloning and expression of a cDNA encoding a catalytically active fragment of calf thymus DNA polymerase alpha

A calf thymus cDNA expression library was constructed in the EcoRI site of lambda gt11 and probed with an antibody raised against calf thymus DNA polymerase alpha. Three classes of antibody-reactive clones were isolated. The largest class carried a 1.9 kilobase calf cDNA insert and expressed a 165-175 kilodalton beta-galactosidase:calf fusion protein which displayed DNA polymerase activity. The characteristic responses of the polymerase activity to alpha-specific inhibitors and antibodies identified the 1.9 kilobase cDNA as a sequence specifically derived from the structural gene encoding the pol alpha catalytic core.     

Peptide models of protein metastable binding sites: competitive kinetics of isomerization and hydrolysis

alpha 2-Macroglobulin and the complement components C3 and C4 each contain a metastable binding site that is essential for covalent attachment. Two cyclic peptides are useful models of these unusual protein sites. Five-membered lactam 1 (CH3CO-Gly-Cys-Gly-Glu-Glp-Asn-NH2) contains an internal residue of pyroglutamic acid (Glp). Fifteen-membered thiolactone 2 (CH3CO-Gly-Cys-Gly-Glu-Glu-Asn-NH2 15-thiolactone) contains a thiol ester bond between Cys-2 and Glu-5. These isomeric hexapeptides are spontaneously interconverted in water. Competing with the two isomerization reactions are three reactions involving hydrolysis of 1 and 2. These five processes were found to occur simultaneously under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C). Best estimates of the five rate constants for these apparent first-order reactions were obtained by comparing the observed molar percentages of peptides 1-4 with those calculated from a set of exponential equations. Both isomerization reactions (ring expansion of 1 to 2, k1 = 6.4 X 10(-5) s-1; ring contraction of 2 to 1, k-1 = 69 X 10(-5) s-1) proceeded faster than any of the hydrolysis reactions: alpha-cleavage of 1 with fragmentation to form dipeptide 3 (k2 = 3.3 X 10(-5) s-1), gamma-cleavage of 1 with ring opening to yield mercapto acid 4 (k3 = 0.35 X 10(-5) s-1), and hydrolysis of 2 with ring opening to give 4 (k4 = 1.9 X 10(-5) s-1). The isomerization rate ratio (k1/k-1 = 10.9) agreed with the isomer ratio at equilibrium (1:2 = 11 starting from 1 and 10 starting from 2). The alpha/gamma regioselectivity ratio (k2/k3 = 9.7) for hydrolysis of the internal Glp residue of 1 was consistent with results for model tripeptides. Part of the chemistry of the protein metastable binding sites can be explained by similar isomerization and hydrolysis reactions.     

Regulatory effects of mast cells on lymphoid cells: the role of histamine type 1 receptors in the interaction between mast cells, helper T cells and natural suppressor cells

We have investigated the role of mast cells as modulators of lymphocyte function because the mast cells are concentrated in the areas of lymphoid storage; they are dependent upon T-cell growth factor for their proliferation; and they appear to be the principle if not sole storage site for histamine. We have tested the influence of mast cells on the proliferation of alloreactive cloned helper T cells, mixed leukocyte reactions, and the suppressive capacity of natural suppressor cells. We used an IL-3-dependent mast cell line that at high numbers (greater than 10(5)) suppressed and at low numbers (10(3) to 6 X 10(4)) augmented the proliferation of TH cells. Addition of histamine to cocultures enhanced the mast cell mediated proliferation of TH cells without directly affecting the helper cells. The action of histamine appeared to be mediated with H1 type receptors on these mast cells. Pretreatment of natural suppressor cells with supernatants from mast cell enhanced their suppressive capability. Here too, histamines enhanced suppression by the NS cell via histamine type 1 receptors on the natural suppressor cells. Our data suggest that mast cells may be a major modulator of the lymphoid cell immune function and demonstrate a role of histamine type 1 receptors in the interaction between mast cells, helper T cells, and natural suppressor cells.     

Peritoneal macrophages exposed to purified macrophage colony-stimulating factor (M-CSF) suppress mitogen- and antigen-stimulated lymphocyte proliferation

The effect of M-CSF-exposed macrophages on murine splenic lymphocyte responses was determined. Resident peritoneal macrophages incubated with purified M-CSF for 48 hr inhibited lymphocyte proliferation to Con A, PHA, and listerial antigen as determined by [3H]TdR uptake, and inhibited Con A-stimulated lymphocyte IL 2 production. The inhibition was similar to that observed with macrophages from BCG-infected mice. Maximal suppression occurred at M-CSF concentrations of 500 U/ml or greater and when the incubation time with M-CSF was 48 hr or more. M-CSF effect was specific because rabbit anti-M-CSF IgG blocked the suppression whereas control rabbit IgG did not. Secretory products of macrophages could not be implicated in this interaction. Catalase and indomethacin, alone or together, did not reverse the inhibition. In addition, putative suppressive factors were not detected in supernatants of M-CSF-stimulated macrophages. Lymphocytes that were removed from macrophage monolayers and were recultured in medium plus Con A were able to proliferate. Macrophages stimulated by M-CSF therefore appear to have inhibitory activity for proliferating lymphocytes, and may play a role in immunoregulatory mechanisms.     

Specific hybridization probes demonstrate fewer xenotropic than mink cell focus-forming murine leukemia virus env-related sequences in DNAs from inbred laboratory mice.

We have derived hybridization probes from analogous 100-base-pair segments located within the N-terminal region of gp70 coding sequences which differentiate xenotropic from mink cell focus-forming (MCF)-related murine leukemia virus (MuLV) DNAs. The MCF probe annealed to the integrated proviruses of all six MCF MuLV isolates tested; the xenotropic probe hybridized to the DNAs of all four xenotropic proviral isolates examined. No cross-hybridization was observed, and neither probe reacted with the env segments of amphotropic or ecotropic MuLV DNAs. Southern blot analysis of HindIII- or EcoRI-digested genomic DNAs from a variety of inbred laboratory mice demonstrated the presence of more MCF- than xenotropic MuLV-related segments in every strain tested.     

Phosphorylation and sulfation of arylsulfatase A accompanies biosynthesis of the enzyme in normal and carcinoma cell lines

Arylsulfatase A (arylsulfate sulfohydrolase, EC 3.1.6.1), a mammalian lysosomal enzyme, is initially synthesized as a 69, 67 and 64 kDa precursor polypeptide in a prostate carcinoma cell line PC-3SF12, in HeLa cells and in a normal human embryonic lung cell line WI-38, respectively. These precursor polypeptides are secreted into the medium or processed to mature enzymes of apparent molecular mass 66, 64 or 62 kDa in PC-3SF12, HeLa or WI-38 cells, respectively. The precursor and mature polypeptides in WI-38 cells are phosphorylated, and the phosphate is lost upon treatment with endo-beta-hexosaminidase H. Arylsulfatase A is also shown to be sulfated in WI-38 cells. The presence of castanospermine, an inhibitor of sulfation of the second N-acetylglucosamine residue of the chitobiose core, does not reduce the extent of sulfation of arylsulfatase A, suggesting that either terminal sugars or the protein is sulfated. Sulfation may have a protective function similar to that of terminal sialic acid residues in glycoproteins. Although the subcellular location of arylsulfatase A is identical in PC-3SF12 and in WI-38 cells, pulse-chase experiments indicate that arylsulfatase A protein has a slower turnover in the prostate carcinoma cell line than it does in the normal human lung cell line. The differences in the apparent molecular weights of arylsulfatase A in the normal and carcinoma cell lines are shown to be due to variations in the carbohydrate content of the enzyme. The apparent molecular mass of the polypeptide chain obtained after endo-beta-hexosaminidase H treatment is 59 kDa, a value which is identical for all three cell lines studied here. These results suggest the possibility of an enhanced activity of terminal glucosyltransferase enzymes in carcinoma cell lines and in tumor tissues. Arylsulfatase A may be a useful marker for studying transformation-related processes in human cell lines.