SIALYLTRANSFERASES IN RAT BRAIN: REACTION KINETICS, PRODUCT ANALYSES, AND MULTIPLICITIES OF ENZYME SPECIES

Abstract— The incorporation of NeuNAc from CMP-NeuNAc into endogenous glycolipids and glyco-proteins, and exogenously added GM_1a (monosialoganglioside) and desialylated fetuin (DS-fetuin) was studied with particulate preparations from 11 to 15 day old rat cerebra. The apparent +K++_m values of the enzyme systems for the different substrates, assayed with 0.5 mg enzyme protein, were: CMP-NeuNAc, 0.13 mm (same with endogenous and exogenous glycolipid and glycoprotein substrates); GM_1a, 0.20 mm ; DS-fetuin, 0.15 mm (or 1.2 mm in terms of acceptor sites). The activities, expressed as nmoles NeuNAc incorporated per 0.5 mg enzyme protein per 30 min incubation at 37°C and pH 6.3, were 0.094, 0.039, 0.17 and 0.64 with the endogenous glycolipids, endogenous glycoproteins, exogenous GM_1a and exogenous DS-fetuin, respectively. Incorporation into endogenous glycolipids was mainly in GM₃, while exogenously added GM_1a was converted to GD_1a. Incorporation into endogenous glycoproteins yields about 20 sialoglycopolypeptides on SDS-polyacrylamide gel electrophoresis. Neura-minidase pretreatment of the particulate enzyme preparation decreased sialylation of the higher molecule weight polypeptides but increased sialylation of the lower molecule weight species. The sialyltransferase activity with the endogenous glycolipid substrates was more heat resistant than the activities with exogenous GM_1a. Since more than 60% of the endogenous glycolipid activity was due to the conversion of lactosylceramide to GM₃, the sialyltransferase responsible for this reaction appears to be different from the one that acts on GM_1a. This was supported by the observation that exogenously added GM_1a did not diminish the incorporation of NeuNAc into endogenous lactosylceramide. These two glycolipid sialyltransferase activities were distinguishable from the glycoprotein sialyltransferase activity since exogenous DS-fetuin did not compete with either the endogenous or the exogenous glycolipids for CMP-NeuNAc.     

SIALYLTRANSFERASES IN RAT BRAIN: INTRACELLULAR LOCALIZATION AND SOME MEMBRANE PROPERTIES

Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.
The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non-ionic detergent mixture, Triton CF-54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM_1a, 700; exogenous DS-fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza-tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.     

Cellular and extracellular siderophores of Aspergillus nidulans and Penicillium chrysogenum.

Aspergillus nidulans and Penicillium chrysogenum produce specific cellular siderophores in addition to the well-known siderophores of the culture medium. Since this was found previously in Neurospora crassa, it is probably generally true for filamentous ascomycetes. The cellular siderophore of A. nidulans is ferricrocin; that of P. chrysogenum is ferrichrome. A. nidulans also contains triacetylfusigen, a siderophore without apparent biological activity. Conidia of both species lose siderophores at high salt concentrations and become siderophore dependent. This has also been found in N. crassa, where lowering of the water activity has been shown to be the causal factor. We used an assay procedure based on this dependency to reexamine the extracellular siderophores of these species. During rapid mycelial growth, both A. nidulans and P. chrysogenum produced two highly active, unidentified siderophores which were later replaced by a less active or inactive product--coprogen in the case of P. chrysogenum and triacetylfusigen in the case of A. nidulans. N. crassa secreted coprogen only. Fungal siderophore metabolism is varied and complex.     

Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas.

A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Concentration of plasminogen activators and inhibitor in the human endometrium at different phases of the menstrual cycle.

The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI-1) have been determined in endometrial curettings obtained from 46 subfertile women during proliferative, early or late secretory phases of the menstrual cycle. t-PA activity and antigen concentrations was significantly higher (P < 0.001) in late secretory endometrium than in proliferative or early secretory endometrium. Higher concentrations of PAI-1 antigen (P < 0.05) were also noted in late secretory phase than in proliferative and early secretory endometrium. However, u-PA concentration was not significantly different and no PAI activity could be demonstrated in the menstrual phases studied. Zymography studies confirmed the presence of both t-PA and u-PA in the endometrium. Ovarian hormonal patterns may therefore influence the activity of plasminogen activators especially of t-PA in the endometrium during various phases of the menstrual cycle.     

Immunologic memory to phosphocholine keyhole limpet hemocyanin. Recurrent mutations in the lambda 1 light chain increase affinity for antigen.

Anti-phosphocholine (PC)-keyhole limpet hemacyanin hybridomas representative of a memory response that express the lambda 1 L chain isotype have a high reactivity to PC-protein. A common feature of these hybridomas possessing high affinity for PC-protein is the occurrence of somatic mutations resulting in replacement changes in three CDR2 positions of the lambda 1 L chain. The influence of each of these three positions on the Ag binding properties of these antibodies was examined by site-specific mutagenesis and expression of recombinant antibody molecules by transfected cells. Affinity measurements and fine specificity profile determinations demonstrated the importance of the three lambda 1 CDR2 positions in Ag binding. Compared to antibodies expressing germline lambda 1, including one with an additional junctional serine that is not encoded by V or J, those antibodies possessing critical changes in CDR2 would have a strong selective advantage based on affinity differences for Ag. Sequence analysis of a group of clonally related hybridomas expressing mutated lambda 1 genes allowed construction of a hypothetical genealogic tree that suggests selection based on changes in CDR2 of lambda 1 in the absence of H chain mutations. The results are consistent with stepwise acquisition of mutations and selection based on affinity constraints.     

Identification and reconstitution of the nucleoside transporter of CEM human leukemia cells

The major nucleoside transporter of the human T leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR). The photolabeled protein migrates on SDS-PAGE gels as a broad band with a mean apparent molecular weight (75,000 +/- 3000) significantly higher than that reported for the nucleoside transporter in human erythrocytes (55,000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However, after treatment with endoglycosidase F to remove carbohydrate, the NBMPR-binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47,000 +/- 3000) identical to that reported for the deglycosylated protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the NBMPR-sensitive nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The NBMPR-binding protein from CEM cells has been solubilized with 1% octyl glucoside and reconstituted into phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of uridine transport activity was achieved using a sonication interval of 5 to 10 s and lipid to protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the protein concentration and was inhibited by NBMPR. Omission of lipid or protein, or substitution of a protein extract prepared from a nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no uridine transport activity. The initial rate of uridine transport, in the vesicles prepared with CEM protein, was saturable with a Km of 103 +/- 11 microM and was inhibited by adenosine, thymidine and cytidine. The Km for uridine and the potency of the other nucleosides as inhibitors of uridine transport (adenosine greater than thymidine greater than cytidine) were similar to intact cells. Thus, although the nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical NBMPR-sensitive nucleoside transport activity both in the intact cell and when reconstituted into phospholipid vesicles.     

Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414

The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). C. thermocellum cellulase activity differed considerably from that of T. reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or β-xylosidase activity; long-chain oligosaccharides instead of short-chain oligosaccharides as initial (15-min) hydrolytic products on microcrystalline cellulose; mainly cellobiose or xylobiose as long-term (24-h) hydrolysis products of Avicel and MN300 or xylan; and high activity and stability at 60 to 70°C. Under optimized reaction conditions, the kinetic properties (V_max, 0.4 μmol/min per mg of protein; energy of activation, 33 kJ; temperature coefficient, 1.8) of C. thermocellum cellulose-solubilizing activity were comparable to those reported for T. reesei, except that the dyed Avicel concentration at half-maximal velocity was twofold higher (182 μM). The cellulose-solubilizing activity of the two crude cellulases differed considerably in response to various enzyme inhibitors. Most notably, Ag²⁺ and Hg²⁺ effectively inhibited C. thermocellum but not T. reesei cellulase at <20 μM, whereas Ca²⁺, Mg²⁺, and Mn²⁺ inhibited T. reesei but not C. thermocellum cellulase at >10 mM. Both enzymes were inhibited by Cu²⁺ (>20 mM), Zn²⁺ (>1.0 mM), and ethylene glycol-bis(β-aminoethyl ether)- N,N-tetraacetic acid (>10 mM). T. reesei but not C. thermocellum cellulose-solubilizing activity was 20% inhibited by glucose (73 mM) and cellobiose (29 mM). Both cellulases preferentially cleaved the internal glycosidic bonds of cellooligosaccharides. The overall rates of cellooligosaccharide degradation were higher for T. reesei than for C. thermocellum cellulase, except that the rates of conversion of cellohexaose to cellotriose were equivalent.