Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver

A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The Km for the substrate at pH 8.0 was 0.3 mM. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca2+, Mg2+, and Ba2+ had essentially no effect, whereas Mn2+, Ni2+, and Cu2+ were inhibitory and Co2+ stimulated activity at low concentrations but inhibited above 5 mM. An increase in the ionic strength of the reaction mixture to 0.3 M decreased the activity by 40%.     

Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis.

We have studied the post-translational processing of p21ras proteins. The primary translation product pro-p21 is cytosolic and is rapidly converted to a cytosolic form (c-p21) of higher mobility on SDS-PAGE. c-p21 is converted in turn to the membrane-bound mature palmitoylated form (m-p21) of slightly higher mobility. These processing steps are accompanied by increases in isoelectric point and in hydrophobicity as judged by Triton X-114 partitioning. Although the increases in electrophoretic mobility and hydrophobicity precede acylation we show that mutation of Cys186, which has been shown to block acylation, also abolishes the pro-p21 to c-p21 conversion. Thus the Cys186 residue is involved in the processing steps prior to acylation. We have identified two processing events which contribute to the pro-p21 conversion. Site-directed mutagenesis to insert tryptophan, which is not present in the wild type, followed by metabolic labelling with [3H]tryptophan has allowed us to map a proteolytic processing event which removes the three C-terminal residues. In addition, both the c-p21 and m-p21 forms are carboxyl-methylated. Approximately one methyl group is incorporated per molecule of p21 at steady state, which can partially account for the increase in isoelectric point. Unlike palmitate, methyl group turnover is not observed.     

Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication.     

Uptake and degradation of hyaluronan in lymphatic tissue.

Afferent lymph vessels entering popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan of high Mr (4.3 x 10(6)-5.5 x 10(6)) and low Mr (1.5 x 10(5)). Analysis of efferent lymph and of residues in the nodes showed that hyaluronan presented by this route is taken up and degraded by lymphatic tissue. Labelled residues isolated in node extracts by gel chromatography and h.p.l.c. included N-acetylglucosamine, acetate, water and a fraction provisionally identified as N-acetylglucosamine 6-phosphate. Between 48 and 75% of the infused material was unrecovered, and had been presumably eliminated through the bloodstream as diffusible residues. Rates of degradation reached as high as 43 micrograms/h in a node of 2 g wt. infused with 56 micrograms/h. Some HA passed into efferent lymph and some was detected in the nodes, but fractions of Mr greater than 1 x 10(6) were not found in either. It is concluded that the amounts and Mr values of hyaluronan released from the tissues into peripheral lymph can be significantly underestimated by analysis of efferent lymph, i.e. lymph that has passed through lymph nodes. A substantial role in the normal metabolic turnover of at least one major constituent of intercellular matrix and connective tissue may now be added to the established functions of the lymphatic system.     

Control of myogenesis in the mouse myogenic C2 cell line by medium composition and by insulin: Characterization of permissive and inducible C2 myoblasts

Using subcloning and manipulations of culture conditions we have isolated from the mouse myogenic cell line C2 a variant cell line that we named inducible. Unlike the progenitor cells that are referred to as permissive, inducible myoblasts differentiate poorly in Dulbecco modified Eagle medium plus fetal calf serum (FCS) and require the presence of insulin at a high concentration (1.6 10(-6) M) or insulin-like growth factor I (IGFI) at a lower concentration (2.5 10(-8) M) to differentiate. Permissive and inducible myoblasts fail to differentiate when grown in MCDB202 medium plus 20% FCS, even after a prolonged arrest in G1 phase. This shows that an arrest in G1 is in itself insufficient to trigger terminal differentiation. Both cell types also exhibit distinct patterns of accumulation of muscle mRNAs corresponding to sarcomeric actins and myosin light chain MLC1A. The possibility that these two cell lines might represent two different stages of the progression of myoblasts toward terminal differentiation is discussed.     

Morphological studies on endocytosis of chondroitin sulphate proteoglycan by rat liver endothelial cells

Summary Endocytosis via the hyaluronic acid/chondroitin sulphate receptor of rat liver endothelial cells was studied ultrastructurally, by use of a probe consisting of chondroitin sulphate proteoglycan attached to 15-nm gold particles. The probe bound to the surface of the cells exclusively in coated regions of the plasma membrane. Internalization at 37° C took place in less than one minute during which time interval the bound probe was transferred to coated vesicles. Further transfer to lysosomes was delayed in association with an accumulation of probes in a prelysosomal compartment consisting of large vacuoles in which probes lined the inner aspect of the membrane. Transport to lysosomes occurred only after a lag phase of at least 40–60 min at 37° C.Abbreviations CS chondroitin sulphate - CSPG chondroitin sulphate proteoglycan - CSPG-Au CSPG-gold complex - EM electronmicroscopical or electron microscopy - HA hyaluronic acid - KC Kuppfer cells - LEC liver endothelial cells - PC parenchymal cells - RES reticuloendothelial system     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells

The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.