Quantitative determination of sialic acid in the monosialoganglioside, GM1, by the thiobarbituric acid method.

Several methods for the quantitative analysis of sialic acids in glycoconjugates require that the sialic acid glycosidic bond be cleaved prior to assay. The conventional (L. Svennerholm, (1958) Acta Chem. Scand. 12, 547-554) procedure for the hydrolysis of complex carbohydrates such as gangliosides employs 0.1 n H+ at 80 degrees C for 60 min. Under these conditions, we find that the monosialoganglioside (GM1) yields less than 50% of the total sialic acid. However, 90% recovery of sialic acid was achieved by supplementing the hydrolysis mixture with 0.2% sodium dodecylsulfate (SDS) and increasing the temperature to 85 degrees C. During hydrolysis under these conditions, N-acetylneuraminic acid (NAN) is degraded at about 7% per hour. If the analytical values for GM1 are corrected for degradation, the recovery of NAN is essentially quantitative.

Protein assay by Coomassie brilliant blue G-250-binding method is unsuitable for plant tissues rich in phenols and phenolases.

Protein estimation in crude homogenates of plant tissues rich in phenols and phenolases was carried out by the dye-binding and, with recommended cautions, by the Lowry et al. methods and the two were compared. The dye-binding method gave grossly erroneous results with a high degree of variation when the homogenizing media differed; this was not due either to the interference by the components of the homogenizing media or to any shift in the absorbance maximum. While the reduced form of the "derived" polyphenolic compounds, generated during tissue homogenization, appeared to enhance dye binding with bovine serum albumin, their influence on the protein assay directly in crude homogenates was extremely diverse. Tissue homogenization in the absence of a reducing agent results in polyquinone-protein complexes which prevent optimal dye binding, resulting in low protein values, while the endogenous phenolics in a homogenate prepared in a mixture of cysteine and NaCl appear to suppress dye-protein complex formation. It is therefore our opinion that the dye-binding method is unsuitable for protein assay in phenol- and phenolase-rich plant tissues.

Phosphoproteins and the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus salivarius. Detection of two different ATP-dependent phosphorylations of the phosphocarrier protein HPr.

Phosphoproteins which arise from incubation of Streptococcus salivarius ATCC25975 crude extracts with [32P]phosphoenolpyruvate and [gamma-32P]ATP, were separated and detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. These procedures were carried out using the methodology that has been developed to allow for the detection of phosphoproteins containing 1-P-histidinyl and 3-P-histidinyl residues, and also to distinguish between these and phosphoproteins containing acid-stable phosphoamino acids such as phosphoserine, phosphothreonine, and phosphotyrosine. Extracts of cells which had been grown with various sugars as carbon sources were investigated to determine both constitutive and inducible phosphoproteins. No evidence was found for phosphoproteins specifically induced by a sugar, and in particular no evidence was found for any IIIsugar phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Incubation with [gamma-32P]ATP showed that histidine-containing phosphocarrier protein (HPr) of the PTS could be phosphorylated to give both acid-stable and acid-labile phosphoamino acid residues. The acid-labile ATP-dependent phosphorylation activity was activated by glucose-6-P and appeared to produce a 3-P-histidinyl residue in HPr.

Isoelectrophoretic separation and the detection of soluble proteins containing acid-labile phosphate: use of the phosphoenolpyruvate:sugar phosphotransferase system as a model system for N1-P-histidine- and N3-P-histidine-containing proteins.

Procedures have been developed for the detection of acid-labile phosphorylations of proteins. The phosphoproteins were separated by native isoelectric focusing while maintaining the gel at about 0 degree C, and denaturing urea-Nonidet isoelectric focusing gels were adapted to run at -10 degrees C. The proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), HPr, which contains 1-P-histidine, and factor IIIglc and enzyme I, which contain 3-P-histidine when they are phosphorylated, were used to develop the conditions. Autoradiography of [32P]-labeled phosphoproteins was carried out on frozen gels which had not been acid fixed in order to avoid hydrolysis of the phosphohistidines . The frozen gels were subsequently fixed and stained, and reautoradiography revealed whether the phosphoproteins were acid stable or labile. In addition to the known proteins of the PTS, at least one other protein whose phosphorylation was dependent on enzyme I and HPr was found in Salmonella typhimurium and Escherichia coli [E.B. Waygood , and R.L. Mattoo (1983) Canad . J. Biochem. Cell Biol. 61, 150-153]. Initial experiments with rat tissues have demonstrated acid-labile phosphorylations in proteins which were either [gamma-32P]ATP or [32P]phosphoenolpyruvate dependent. The interconversion of phosphoenolpyruvate and ATP in crude extracts of bacterial cells was examined, and appropriate controls were found. Protein phosphorylation dependent upon phosphoenolpyruvate was much greater in S. typhimurium and E. coli than the corresponding ATP-dependent phosphorylation, while the opposite was found for rat tissues.

Activation of phosphoenolpyruvate-dependent protein kinase by cytidine 5'-triphosphate in rat skeletal muscle.

The effects of nucleotides on phosphoenolpyruvate-dependent protein kinase in rat skeletal muscle were examined. Various ribonucleotide triphosphates were tested and the protein kinase reaction was maximally activated by cytidine 5'-triphosphate followed by uridine 5'-triphosphate and guanosine 5'-triphosphate. No activation by adenosine 5'-triphosphate was observed. Cytidine 5'-diphosphate also activated the reaction but to a significantly lesser extent. Cytidine 5'-monophosphate and cyclic cytidine 3',5'-monophosphate showed no effect whereas cytidine was slightly inhibitory.

Phosphoproteins and the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIImannose, IIIfructose, IIIglucitol, and the phosphorylation of enzyme IImannitol and enzyme IIN-acetylglucosamine.

Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [32P]phosphoenolpyruvate or [gamma 32P]ATP have been resolved and detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Simple techniques were found such that distinctions could be made between phosphoproteins containing acid-labile or stable phosphoamino acids and between N1-P-histidine and N3-P-histidine. Phosphoproteins were found to be primarily formed from phosphoenolpyruvate, but because of an efficient phosphoexchange, ATP also led to the formation of the major phosphoenolpyruvate-dependent phosphoproteins. These proteins had the following apparent subunit molecular weights: 65,000, 65,000, 62,000, 48,000, 40,000, 33,000, 25,000, 20,000, 14,000, 13,000, 9,000, 8,000. Major ATP-dependent phosphoproteins were detected with apparent subunit molecular weights of 75,000, 46,000, 30,000, and 15,000. Other minor phosphoproteins were detected. The phosphorylation of the 48,000- and 25,000-MW proteins by phosphoenolpyruvate was independent of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme IIN-acetylglucosamine (membrane bound; MW = 65,000); enzyme IImannitol (membrane bound; MW = 62,000); IIIfructose (soluble; MW = 40,000); IIImannose (partially membrane associated; MW = 33,000); IIIglucose (soluble; MW = 20,000); IIIglucitol (soluble; MW = 13-14,000); HPr (soluble; MW = 9,000); FPr (fructose induced HPr-like protein (soluble; MW = 8,000). HPr and FPr are phosphorylated on the N-1 position of a histidyl residue while all the others appear to be phosphorylated on an N-3 position of a histidyl residue. These studies identify some previously unknown proteins of the PTS and show the phosphorylation of others, which although previously known, had not been shown to be phosphoproteins.

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle.

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.

A novel phosphoprotein dependent on the bacterial phosphoenolpyruvate-sugar phosphotransferase system.

A protein has been fond by isoelectricfocusing and autoradiography in Escherichia coli and Salmonella typhimurium which was phosphorylated by enzyme I and an histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate-sugar phosphotransferase system (PTS). This protein was not factor III glc nor was it specifically induced by fructose. Its presence in soluble crude extracts was dependent upon growth conditions; however, the two bacteria had different patterns and amounts in respect to this novel protein. The protein was present in S. typhimurium SB2950 which has an extensive deletion through the pts operon, thus indicating that it must be coded for elsewhere on the genome.

Determination of the levels of HPr and enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium.

The levels of histidine-containing protein HPr and enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system of Escherichia coli strains 1100, NC3, W3110, and P650 and Salmonella typhimurium strains SB3507 and LJ144 have been determined by quantitative sugar phosphorylation assay and immunochemically. The levels have been determined for cells grown on minimal salts with glucose, fructose, mannitol, glycerol, and lactate and on nutrient broth. All determinations indicate a two- to three-fold change in the levels of enzyme I and HPr between growth on hexoses, which gave the higher levels, and the other growth substrates. The highest levels were not always found in glucose-grown cells. Antibodies were produced in rabbits using purified proteins from E. coli P650. The activity measurements and immunochemically determined enzyme I protein gave specific activities in the crude extracts of E. coli strains which were similar to that of the pure enzyme. The wild-type S. typhimurium enzyme I in crude extracts did not have the same immunochemical reactivity, although there was a considerable cross-reaction and the specific activity appeared to be half that of pure enzyme I. The HPr from both E. coli and S. typhimurium behaved identically and, although the immunoprecipitation was weak, it did indicate that HPr assays may not be as reliable as the enzyme I assays. The relative amounts of enzyme I and HPr found indicate that there are between 10- and 20-fold more HPr molecules in a cell than enzyme I subunits which form active dimers.

Studies on Sequential Parasitism by Orobanche and Cuscuta on Petunia hybrida: Choline Kinase and Phospholipid.

Parasitism by Cuscuta and Orobanche on Petunia hybrida resulted in decreased choline kinase activity and phospholipids in the host shoots. The Cuscuta-infected host roots suffered a decline in phospholipid concentration with no appreciable change in enzyme activity, whereas the roots of the Orobanche-infected plants exhibited a substantial increase in phospholipid concentration despite a marked lowering in enzymic activity. Superimposition of infection by Cuscuta on Orobanche-infected plants resulted in an increase in both enzyme activity and phospholipid in host shoots; the host roots recorded a decline in phospholipid, although enzyme activity was increased. As compared to the filaments infecting singly, Cuscuta, in sequential infection, registered an increase in phospholipid concomitant with a fall in enzyme activity, whereas the root parasite revealed a lowered enzyme activity and a slight decrease in phospholipid. It is hypothesized that a physiological response to infection by root parasite was an accumulation of phospholipids at the region under infection, and to that by shoot parasite was an uptake of phospholipids by the parasite from the host; this was effected not by de novo synthesis but rather by mobilization from distal regions.