Unusual heterogeneity in the glycosylation of the G protein of the hazelhurst strain of vesicular stomatitis virus

The asparagine-linked oligosaccharides of the G protein of the Hazelhurst subtype of the New Jersey serotype of vesicular stomatitis virus (VSV) have been compared with the oligosaccharides from the G protein of the well-characterized Indiana serotype of VSV, with baby hamster kidney cells in monolayer culture as the host for both viruses. [3H]Glucosamine- and [3H]mannose-labeled glycopeptides from the G protein of purified virus were analyzed by the combined techniques of endo-beta-N-acetylglucosaminidase H (ENDO-H) digestion, concanavalin A and lentil lectin affinity chromatography, and Bio-Gel P-4 chromatography. Although almost all of the Indiana G protein oligosaccharides were acidic-type structures, as expected from previous studies; the Hazelhurst G protein contained a mixture of acidic-type, hybrid-type containing sialic acid, and neutral-type (predominantly Man5-6GlcNAc2-Asn) structures. The vast majority of acidic-type oligosaccharides from both the Hazelhurst and Indiana G proteins were diantennary structures, with less than half containing fucose linked to the innermost N-acetylglucosamine. Additional analysis of the Hazelhurst G protein by ENDO-H digestion and gel electrophoresis suggested that some of the mature G polypeptides contained acidic-type structures at both glycosylation sites, whereas the remainder contained an ENDO-H-resistant, acidic-type structure at one site and an ENDO-H-sensitive, hybrid- or neutral-type structure at the other site.     

Induction of fructose 1,6-bisphosphatase and glucose 6-phosphatase in fetal mouse liver

Fructose 1,6-bisphosphatase and glucose 6-phosphatase were induced in organ cultures of liver tissues from 15- and 19-day-old fetal mice, using a culture method that allowed the tissues to be maintained for 7 days in the absence of serum. In cultures from 15-day-old fetal liver, both enzyme activities increased significantly per milligram of DNA after a lag period of 1 to 3 days. In cultures from 19-day-old fetal liver only glucose 6-phosphatase increased in the absence of inducer. N6,O2'-Dibutyryladenosine 3',5'-monophosphate enhanced the rate of increase in fructose 1,6-bisphosphatase and glucose 6-phosphatase activities. The minimum effective concentration of the cyclic nucleotide was approximately 10(-6) M. Dexamethazone inhibited the increase in fructose 1,6-bisphosphatase during culture for 7 days. Glucose 6-phosphatase activity was enhanced by dexamethazone in cultures from 19-day-old fetal liver, but was without effect on glucose 6-phosphatase in cultures from 15-day-old fetal liver. The minimum inhibitory concentration of dexamethazone was less than 10(-8) M. The results suggest a complicated effect of the cyclic nucleotide on the two enzymes in fetal mouse liver as well as different mechanisms of action of dexamethazone on the induction of two enzymes.     

Biosynthesis of lutropin in ovine pituitary slices: Incorporation of [35S]sulfate in carbohydrate units

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H₂³⁵SO₄. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the α and β subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [³⁵S]sulfate was also incorporated into several other proteins in addition to LH. The location of ³⁵SO₄^2? in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[³⁵S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[³⁵S]sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of ³⁵SO₄^2? by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.     

Surface-charge related actions of polylysine on thylakoid membranes

Polycation binding to the negatively charged surface of chloroplast thylakoid membranes is known to cause an inhibition of photosystem I activity. It also interferes with the cation-dependent rearrangement of chlorophyll proteins in the thylakoid membrane. It was shown that added anions prevented or reversed the inhibition of photosystem I by polylysine without decreasing its binding to the membranes. Anions also caused a change in the interaction of the chlorophyll proteins in polylysine-treated thylakoids as indicated by an increase in the relative fluorescence intensity from photosystem II. In both cases, the relative effectiveness of the anions tested depended on their valence; for example, the tetravalent species Fe(CN)₆^4t- was effective at a concentration at least 2 orders of magnitude lower than the divalent species SO₄^2?. These results suggest that anions act by screening the positive charge of the polylysine-coated membrane surface. Measurements of the response of the anionic fluorescent probe 1-anilinonapthalene-8-sulfonate to an addition of anions to polylysine-treated thylakoids supported this contention. It was concluded that the action of polylysine on photosystem I and on the chlorophyll proteins is mediated by changes of the electrical properties of the thylakoid membrane and may not involve a direct binding of the polycation to the affected membrane proteins.     

A kinetic and spectral study of the alkaline transitions of chloroperoxidase

The optical spectrum of chloroperoxidase in the near ultraviolet and visible region was studied from pH 6 to 12. Chloroperoxidase undergoes a first transition which is irreversible at pH 7 and a second transition near pH 11. The second transition is reversible provided the incubation period above pH 11 is kept as short as possible. The spectral properties of the intermediates were studied in the Soret region by means of a rapid scan apparatus. The rates of the transitions were measured in a stopped-flow apparatus. The pH dependence of both the spectra and the rate constants indicate that at least three ionizations are involved in the first alkaline transition.     

The conformational analysis of oligosaccharides by H-NMR and HSEA calculation

The application of 1H-nuclear Overhauser enhancement, 1H-spin-lattice-relaxation-time and 1H-chemical shift measurements for the assessment of the conformational preferences of oligosaccharides are briefly reviewed. It is demonstrated that additivity rules, for the correlation of the chemical shifts of similar hydrogen atoms in different oligosaccharides, can be useful in the conformational analysis of oligosaccharides when the differential chemical shifts are greater than 0.1 ppm. These often can be attributed to specific interunit deshielding of a hydrogen atom by an oxygen atom with which it is in strong nonbonded interaction. HSEA calculations are used to demonstrate that differential chemical shifts of less than 0.1 ppm can have origins that are not significant to the overall conformational preferences of the oligosaccharides which are being compared. Both shielding and deshielding effects can arise from a change in the orientation of a substituent group as the result of the introduction of a sugar on a neighboring unit. It is demonstrated that substituent groups, such as hydroxymethyl and acetamido groups, on occasions, should be treated in HSEA calculations as freely rotating about their linkage to a pyranose ring.     

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.     

Isocitrate lyase: artifacts and multiple enzyme forms

Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.     

A time lag in the onset of ATP-Pi exchange catalyzed by purified ATP-synthase (CF0-CF1) proteoliposomes and by chloroplasts

The time course of ATP-Pi exchange which is catalyzed by the isolated chloroplast ATP synthase in phospholipid vesicles was studied. The following observations were made. (i) The onset of 32Pi incorporation into ATP lags behind ATP hydrolysis. The lag lasts for about 2 min at 37 degrees C and is followed by a steady-state rate which is constant for more than 30 min. Under the same experimental conditions, ATP hydrolysis shows an initial burst followed by a constant, slower rate. (ii) The initial lag is independent of Mg-ATP concentration in the range 0.2-5 mM and of the presence of ADP. In contrast, the steady-state rate of ATP-Pi exchange has an apparent Km of 0.3 mM for Mg-ATP and is stimulated by ADP. (iii) Increasing the temperature from 30 to 45 degrees C decreases the lag from 6 min to zero. The steady-state rate of ATP-Pi exchange is affected to a much smaller extent by the temperature in this range. (iv) The lag is insensitive to valinomycin or tetraphenylboron, while the steady-state rate is partially inhibited. Nigericin and protonophores affect both the lag and steady-state rate. (v) ATP-induced membrane potential formation, as followed by oxonol VI, does not correlate with the lag in its kinetics and temperature dependence. ATP-induced pH gradient formation could not be detected in the proteoliposome system. (vi) Light-triggered ATP-Pi exchange in chloroplasts shows essentially the same time course as the proteoliposome system, but the lag lasts for only about 20 s at room temperature and is unaffected by a preexisting proton gradient. These results suggest that the initial lag in ATP-Pi exchange does not reflect the time required for the buildup of a protomotive force (delta - mu H+) nor the time required to produce ADP. It is suggested, therefore, that the lag reflects an internal autocatalytic conformational change in the ATP-synthase complex which is initiated by ATP hydrolysis and which converts the enzyme from an "exclusive ATPase state" to a "reversible ATP-synthase state". This slow transition is not directly coupled to a trans-membrane pH or potential gradient.     

Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate

Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme. Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited. NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site. The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm. Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme. Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm. These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site. Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain. These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.