Microflora associated with the internal surfaces of rubber and stainless steel milk transfer pipeline

Sterile sections of rubber and stainless steel milk transfer pipeline were inserted sequentially into a milking installation and soiled with fresh raw milk over a period of 5 d. The resultant adherent microbial population was removed and the generic composition of mesophilic and psychotropic types was determined. In all cases Acinetobacter spp. were found to predominate (59.5-75.6%). The generic composition of the raw milk used to soil the milking unit (with inserted pipe section) was determined once during each 5-d soiling period. In general the milk was found to contain a mixed flora in which Gram-positive organisms predominated.     

Purification and characterization of six cytochromes P-450 from hepatic microsomes of immature female rats

Six rat hepatic cytochromes P-450, named P-450IF-1-6, were purified from hepatic microsomes of immature female rats by high-performance liquid chromatography with anion-exchange, cation-exchange, and hydroxylapatite columns. The purified forms, except for P-450IF-4, gave a single protein-staining band on SDS-polyacrylamide gel electrophoresis, with a minimum molecular weight of 50,000 for P-450IF-1, 49,000 for P-450IF-2, 47,000 for P-450IF-3, 53,500 for P-450IF-5, and 54,000 for P-450IF-6. The CO-reduced spectral maximum of these forms was at 450 nm for P-450IF-1, 448 nm for P-450IF-2, 451 nm for P-450IF-3, 449 nm for P-450IF-4, 449 nm for P-450IF-5, and 450 nm for P-450IF-6. All of these cytochromes had the low-spin state of heme in the oxidized form. P-450IF-4 had high metabolic activity for both benzphetamine and 7-ethoxycoumarin. P-450IF-5 had moderate activity toward 7-ethoxycoumarin. P-450IF-3 catalyzed the hydroxylation of testosterone at the 7 alpha-position effectively, but the other forms did not hydroxylate testosterone. Analysis of the NH2-terminal sequence showed that P-450IF-1, 2, 3, 5, and 6 differed structurally from each other. The sequences of P-450IF-1 and IF-2 were somewhat homologous, but the NH2-terminal sequences of the other forms were all different. Based on these results, we concluded that P-450IF-1 corresponded to one of the phenobarbital-inducible forms in male rat liver. P-450IF-2 was a female-specific form and its concentration was low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene

A procedure was developed to purify large quantities of PII protein from an Escherichia coli strain which contains a multicopy plasmid harboring the structural gene of PII (the glnB gene). Ultraviolet spectra of uridylylated and unuridylylated PII were obtained using the purified PII and empirical formulas to calculate the concentration of protein and the average number of uridylylated subunits per molecule were derived. A continuous fluorometric assay for the measurement of uridylylated PII (PIID) and adenylyltransferase (ATase) was also established. Rate measurements at various concentrations of PIID and at a fixed concentration of ATase showed that a tetrameric PIID molecule interacts with only one ATase molecule at a time. The complete nucleotide sequence of the glnB gene was determined and parts of the deduced amino acid sequence were confirmed by the results of amino acid sequence analysis of peptides. The PII subunit consists of 103 amino acids (Mr = 11,580). Two tyrosines reside at positions 46 and 51, where Tyr51 is the site of uridylylation. Nucleotide sequence analysis of the upstream region showed no obvious sites for the binding of RNA polymerase, indicating that the glnB gene is a part of an as yet unidentified operon.     

Interaction of rat mammary gland thioesterase II with fatty acid synthetase is dependent on the presence of acyl chains on the synthetase

The interaction between rat mammary gland thioesterase II and fatty acid synthetase has been studied by a variety of physicochemical techniques. Pyrene-labeled thioesterase II does not exhibit increased fluorescence anisotropy when mixed with fatty acid synthetase, suggesting that the enzymes do not readily form a complex. Nevertheless, the functional interaction between the enzymes can be easily demonstrated by observing the hydrolysis, by unmodified thioesterase II, of acyl chains from their thioester linkage to the 4-phosphopantetheine of the fatty acid synthetase. This hydrolytic reaction is not inhibited even in the presence of a large excess of fatty acid synthetase with vacant 4'-phosphopantetheine thiols, indicating that interaction occurs only between thioesterase and fatty acid synthetase species which carry acyl chains on the 4'-phosphopantetheine thiols. A novel model system was devised which allowed us to explore the nature of the physical interaction between the two enzymes under conditions where the synthetase was actively engaged in acyl chain assembly. Fatty acid synthetase was treated with phenylmethanesulfonyl fluoride to inhibit its resident thioesterase activity, immobilized via a specific antibody to a column of Sepharose 4B, and exposed to the substrates required for acyl-enzyme assembly. When thioesterase II was introduced to the column, it passed through unretarded even though it efficiently catalyzed hydrolysis of the immobilized S-acyl synthetase en route. These results indicate that the two enzymes associate when an acyl chain is present on the synthetase and that they dissociate rapidly following completion of the catalytic process. Thus, the mammary system differs from that of the avian uropygial gland in which the two enzymes associate to form a stable complex even in the absence of substrates.     

Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.     

A growth inhibitory protein secreted by human diploid fibroblasts. Partial purification and characterization

We have identified and partially purified a growth inhibitor protein secreted by human diploid fibroblast cells. This protein is not secreted constitutively but only after induction with the double stranded hetero duplex polyriboinosinic:polyribocytidylic acid. The growth inhibitory activity has been purified 3,800-fold and has an estimated molecular mass of 12,000 daltons. The protein will inhibit the growth in culture of human diploid fibroblast cells, human cells derived from tumors, and mouse L cells. Although interferon-beta is secreted with the growth inhibitory protein, the partially purified growth inhibitory protein has no antiviral activity, and its activity is not neutralized by antibodies to interferon-alpha, interferon-beta, and interferon-gamma. We believe this growth inhibitory activity to reside in a newly defined protein and have named it fibroblast-derived growth inhibitor.     

Spin trap determination of free radical burst kinetics in stimulated neutrophils

Electron spin resonance spin-trapping methods were used to investigate the free radical production kinetics of neutrophils stimulated with phorbol myristate acetate (PMA) and opsonized zymosan (OPZ). Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, the principle spin adduct observed is DMPO-OH (trapped hydroxyl radical). The DMPO-OH ESR signal amplitude was observed to decay exponentially. In such cases a simple method may be used to analyze the raw kinetics amplitude data to yield true production rate and net production data. The method, pitfalls, and self-consistency criteria are illustrated with PMA and OPZ-stimulated neutrophils at 25 and 37 degrees C under varying oxygen tensions, and with noise-free simulated data. The simulations demonstrate that rate results are relatively insensitive to the precise choice of decay time constant, tc, while net production results are very sensitive to the choice of tc used to analyze the raw data. OPZ (0.6-2.4 mg/ml) yields a strong, sharp neutrophil burst which peaks in 2 min or less while PMA yields a slower burst which peaks in 3.4-14 min for PMA concentrations of 500-50 ng/ml, respectively. Increased oxygen tension during the PMA experiments increased the spin adduct lifetime. The methods presented are applicable to other cell systems or spin adducts which exhibit first order decay.     

The three-dimensional structure of acarbose bound to glycogen phosphorylase

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.     

Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis

DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.