A shift in the optimum pH of phospholipase D produced by activating long-chain anions

1. The activity of phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) towards ultrasonically treated phosphatidylcholine or large phosphatidylcholine particles activated with ether was maximal near pH5, and there was little activity above pH6. 2. When the enzyme was activated by the addition of phosphatidic acid to large phosphatidylcholine particles the pH optimum was shifted to pH6.5 irrespective of the amount of activator added. 3. When the enzyme was activated with low concentrations of dodecyl sulphate the pH optimum was 5.5 with little activity above pH6. With higher concentrations of dodecyl sulphate the pH-activity profile was shifted upwards towards a pH optimum of 6.5-6.6, the magnitude of the shift depending on the extent of the hydrolysis. 4. The shifts in the pH-activity profiles cannot be correlated with changes in the ;surface pH' of the substrate particles calculated from the measurement of their zeta-potentials (electrophoretic mobilities).     

Effects of dietary chlortetracycline on the antimicrobial resistance of porcine faecal streptococcaceae

Breeding pigs and one-half of their progeny were fed antimicrobial-free rations; the other half of the progeny received rations supplemented with 100 g of chlortetracycline (Ctc)/ton. Effects of dietary Ctc with respect to the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Diversity of antimicrobial resistance (AMR) patterns and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1140 of 1150 isolates (99%) into 10 groups. Three of these were biotypes of Streptococcus faecium and contained the largest number of isolates ( n = 934, 81%). Streptococcus faecalis, Strep. morbillorum, Pediococcus halophilus and Gemella haemolysans also were isolated. Generally, the proportion of tetracycline-resistant strains for a species or biotype was greater from pigs fed Ctc, although differences were not significant ( P > 0.05). There was a significant difference ( P > 0.05) among all the groups for the percentage of penicillin-resistant strains in a biotype of Strep. faecium. Overall, 57 and 43 different AMR patterns, including 2 to 11 and 1 to 11 resistance determinants, were demonstrated in isolates from control pigs and pigs fed Ctc, respectively. Modal AMR patterns in species and biotypes were the same from both progeny groups, except for Strep. faecium. AMR pattern diversity was decreased for strains from pigs fed Ctc. Similar proportions of resistant strains from each group of progeny pigs were accompanied by decreased AMR pattern diversity in strains from pigs fed Ctc. These results indicated a change in distribution of AMR phenotypical patterns, rather than a change in overall frequency of individual resistant phenotypes.     

Glycopeptide fractions prepared from purified central and peripheral rat myelin

Myelin was purified from rat brain and sciatic nerve after invivo labeling with [³H]fucose and [¹⁴C]glucosamine to provide a radioactive marker for glycoproteins. The glycoproteins in the isolated myelin were digested exhaustively with pronase, and glycopeptides were isolated from the digest by gel filtration on Bio-Gel P-10. The glycopeptides from brain myelin separated into large and small molecular weight fractions, whereas the glycopeptides of sciatic nerve myelin eluted as a single symmetrical peak. The large and small glycopeptide fractions from central myelin and the single glycopeptide fraction from peripheral myelin were analyzed for carbohydrate by colorimetric and gas liquid chromatographic techniques. The glycopeptides from brain myelin contained 2.4 μg of neutral sugar and 0.59 μg of sialic acid per mg total myelin protein, whereas sciatic nerve myelin glycopeptides contained 10 μg of neutral sugar and 3.8 μg of sialic acid per mg total protein. Similarly, the gas-liquid chromatographic analyses showed that the glycopeptides from peripheral myelin contained 4- to 7-fold more of each individual per mg total myelin protein than those from central myelin. Most of the sialic acid and galactose in the glycopeptides from central myelin were in the large molecular weight fraction, and the small molecular weight glycopeptides contained primarily mannose and $\text{N-}\text{acetylglucosamine}$. The considerably higher content of glycoprotein-carbohydrate in peripheral myelin supports the results of gel electrophoretic studies, which indicate that the major protein in peripheral myelin in glycosylated while the glycoproteins in purified central myelin are quantitatevely minor components.     

Effects of dietary chlortetracycline on the antimicrobial resistance of broiler faecal Streptococcaceae

A breeder flock and a control group of progeny birds were fed antimicrobial-free rations; a second group of progeny received rations supplemented with 50 g chlortetracycline (Ctc)/ton. Effects of dietary Ctc on the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Antimicrobial resistance (AMR) pattern diversity and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1321 (97%) of 1360 isolates into eight species or biotypes. The largest cluster ( n = 659, 48%) was a biotype of Streptococcus faecalis. Three clusters were biotypes of Streptococcus faecium and contained 580 isolates (42%). The isolates were susceptible to ampicillin and almost uniformly resistant to methicillin, neomycin, streptomycin, sulfadiazine and tetracycline. There were 54 and 47 different AMR patterns, including 0 to 11 and 1 to 11 resistance determinants, in isolates from control and Ctc-fed birds, respectively. Modal AMR patterns for Strep. faecalis and one biotype of Strep. faecium were very similar for all three groups of birds. However, modal patterns in a second biotype of Strep. faecium varied considerably for all three groups. Interpretation of AMR pattern diversities were equivocal among biotypes from both progeny groups. The variable distribution of isolates, proportions of resistant strains, modal patterns and diversity indices among the progeny were probably due to their exposure to different environmental sources of bacteria.     

The hydrolysis of monolayers of phosphatidyl-[Me-14C]choline by phospholipase D

1. The hydrolysis of monolayers of phosphatidyl[Me-(14)C]choline at the air/water interface by phospholipase D (phosphatidylcholine phosphatidohydrolase) was investigated by a surface-radioactivity technique by using a flow counter. 2. Phosphatidylcholine of high specific radioactivity was prepared biosynthetically in good yield from [Me-(14)C]choline by using Saccharomyces cerevisiae. 3. At initial monolayer pressures between 12 and 25 dynes/cm. the hydrolysis occurred in two stages, an initial slow hydrolysis followed by a rapid hydrolysis. Below 3dynes/cm. and above 28dynes/cm. no enzymic hydrolysis of pure phosphatidylcholine monolayers could be detected. 4. The rapid hydrolysis was proportional to the enzyme concentration in the subphase, its pH optimum was 6.6, and 0.2mm-Ca(2+) was required for maximal activity. 5. Hydrolysis of the film was accompanied by a pronounced fall in the surface pressure even though the phosphatidic acid formed did not leave the film. When the pressure fell to low values the hydrolysis ceased even if the film was only partially hydrolysed. 6. Above monolayer pressures of 28dynes/cm. enzymic hydrolysis could be initiated by inclusion of phosphatidic acid (and less effectively stearyl hydrogen sulphate) in the film, although the rates were not appreciably higher than those observed at 25dynes/cm. with a pure phosphatidylcholine film. 7. The initiation of the hydrolysis by phosphatidic acid was facilitated by the inclusion of high Ca(2+) concentrations and certain carboxylic acid buffer anions in the subphase, although these did not activate by themselves. 8. The initiation of the hydrolysis at high pressures could not be related to any change in the surface potential brought about by the addition of the long-chain anions to the film, nor could it be ascribed to a surface dilution effect. 9. The results are discussed in relation to previous studies on the hydrolysis of phosphatidylcholine particles by the enzyme and also similar investigations on phosphatidylcholine monolayers with other phospholipases.