Purification and properties of milk xanthine dehydrogenase.

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.     

Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.     

Ovarian follicular response of mares to GnRH agonist (leuprolide acetate) treatment after pituitary suppression

Follicular growth and ovulation were monitored in 18 horse mares during a control cycle and during a cycle in which the mares received a GnRH agonist, leuprolide acetate (LA; 200 or 400 mug), twice daily until ovulation. Prior to both of these cycles, follicular growth was suppressed using a 10-day estrogen-progesterone treatment regimen, with prostaglandin F-2alpha (10 mg) administered on Day 10. Four of the mares treated with LA remained anovulatory for at least 3 weeks after the end of treatment and were excluded from statistical analysis. The dosage of LA did not affect response. Treatment with LA significantly (P=0.0375) increased the percentage of large follicles per ovulation (i.e., follicles greater than 30 mm in diameter on the day on which the largest follicle reached 35 mm) and also increased (P=0.0539) the diameter of the second largest follicle. However LA did not significantly alter the number of ovulations. Mean daily concentrations of luteinizing hormone (LH) were not significantly different during treatment and control cycles. The LH in blood samples collected repeatedly on Day 19 after the start of estrogen-progesterone treatment did not show a difference in frequency or amplitude of pulses between treatment and control cycles. Mares were artificially inseminated during estrus and the embryos were recovered. Fewer embryos were recovered per ovulation from mares after treatment with LA (26%) than during the control cycle (64%). Results indicate that treatment with LA either suppressed follicular activity or induced multiple follicular growth.     

Evidence for domain organization within the 61-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain.

The complete amino acid sequence of the 61-kDa calmodulin-dependent, cyclic nucleotide phosphodiesterase (CaM-PDE) from bovine brain has been determined. The native protein is a homodimer of N alpha-acetylated, 529-residue polypeptide chains, each of which has a calculated molecular weight of 60,755. The structural organization of this CaM-PDE has been investigated with use of limited proteolysis and synthetic peptide analogues. A site capable of interacting with CaM has been identified, and the position of the catalytic domain has been mapped. A fully active, CaM-independent fragment (Mr = 36,000), produced by limited tryptic cleavage in the absence of CaM, represents a functional catalytic domain. N-Terminal sequence and size indicate that this 36-kDa fragment is comprised of residues 136 to approximately 450 of the CaM-PDE. This catalytic domain encompasses a approximately 250 residue sequence that is conserved among PDE isozymes of diverse size, phylogeny, and function. CaM-PDE and its PDE homologues comprise a unique family of proteins, each having a catalytic domain that evolved from a common progenitor. A search of the sequence for potential CaM-binding sites revealed only one 15-residue segment with both a net positive charge and the ability to form an amphiphilic alpha-helix. Peptide analogues that include this amphiphilic segment were synthesized. Each was found to inhibit the CaM-dependent activation of the enzyme and to bind directly to CaM with high affinity in a calcium-dependent manner. This site is among the sequences cleaved from a 45-kDa chymotryptic fragment that has the complete catalytic domain but no longer binds CaM. These results indicate that residues located between position 23 and 41 of the native enzyme contribute significantly to the binding of CaM although the involvement of residues from additional sites is not excluded.     

Free radical damage to proteins: the influence of the relative localization of radical generation, antioxidants, and target proteins

Free radicals were generated at known rates in the aqueous phase (by means of 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) and in a membranous (lipid) phase (by means of 2,2'-azobis (2,4-dimethylvaleronitrile [AMVN]). A soluble protein (bovine serum albumin: BSA), and membranes of lysed mitochondria containing radioactively labeled monoamine oxidase (MAO), were exposed to the resultant radical fluxes. Antioxidants were added to the system, either in the aqueous phase (Trolox) or in a liposomal membrane phase (alpha-tocopherol). Protein damage was assessed as tryptophan oxidation and conformational changes in tryptophan fluorescence of the soluble protein, BSA, and as fragmentation of both BSA and monoamine oxidase. Radicals generated in the aqueous phase, by AAPH, were effective in damaging BSA and MAO. Radicals generated within the liposome membrane phase (by AMVN) were less effective against BSA than those deriving from AAPH. Liposomal AMVN radicals could damage MAO, present in a separate membranous phase, though again, less effectively than could AAPH-derived radicals. BSA could be protected by Trolox, the aqueous soluble antioxidant, but hardly by tocopherol itself. Damage to MAO was limited by Trolox, and also by the hydrophobic antioxidant, tocopherol. Damaging reactions due to radicals generated in a membrane phase were significantly accelerated when the membrane was peroxidizable (soybean phosphatidylcholine) rather than nonperoxidizable (saturated dimyristoyl phosphatidylcholine). Thus lipid radicals also played some role in protein damage in these systems. BSA was attacked similarly in the presence or absence of liposomes by AAPH. Correspondingly, BSA could inhibit the peroxidation of liposomes induced by AAPH and less efficiently that induced by AMVN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of O-antigen distribution in lipopolysaccharide.

O-antigen units are nonuniformly distributed among lipid A-core molecules in lipopolysaccharide (LPS) from gram-negative bacteria, as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the actual distribution patterns are complex, multimodal, and strain specific. Although the basic biochemical steps involved in synthesis and polymerization of O-antigen monomers and their subsequent attachment to lipid A-core are known, the mechanism by which specific multimodal distribution patterns are attained in mature LPS has not been previously considered theoretically or experimentally. We have developed probability equations which completely describe O-antigen distribution among lipid A-core molecules in terms of the probability of finding a nascent polymer (O antigen linked to carrier lipid) of length k (Tk) and the probability that a nascent polymer of length k will be extended to k + 1 by polymerase (pk) or transferred to lipid A-core by ligase (qk). These equations were used to show that multimodal distribution patterns in mature LPS cannot be produced if all pk are equal to p and all qk are equal to q, conditions which indicate a lack of selectivity of polymerase and ligase, respectively, for nascent O-antigen chain lengths. A completely stochastic model (pk = p, qk = q) of O-antigen polymerization and transfer to lipid A-core was also inconsistent with observed effects of mutations which resulted in partial inhibition of O-antigen monomer synthesis, lipid A-core synthesis, or ligase activity. The simplest explanation compatible with experimental observations is that polymerase or ligase, or perhaps both, have specificity for certain O-antigen chain lengths during biosynthesis of LPS. Our mathematical model indicates selectively probably was associated with the polymerase reaction. Although one may argue for a multimodal distribution pattern based on a kinetic mechanism i.e., varying reaction parameters in space or in time during cell growth, such a model requires complex sensory and regulatory mechanisms to explain the mutant data and mechanisms for sequestering specific components of LPS biosynthesis to explain the distribution pattern in normal cells. We favor the simple alternative of enzyme specificity and present generalized equations which should be useful in analysis of other analogous biochemical systems.     

31P- and 1H-NMR investigations of the effect of n-alcohols on the hydrolysis by phospholipase A2 of phospholipid vesicular membranes

31P- and 1H-NMR spectroscopy of small, unilamellar egg yolk phosphatidylcholine (PC) vesicles in the presence of the lanthanide ion Dy3+ have been used to study the effect of various n-alcohols on the permeability induced by the action of the enzyme phospholipase A2 (PLA2). The method allows the monitoring of the number of PC and lysoPC molecules in the outer and inner monolayers. The results indicate that the initial rate of hydrolysis of PC by PLA2 is increased by all the n-alcohols but in a chain-length dependent manner and that the maximum rate occurs at n = 8 (octan-1-ol). The subsequent rate is dependent upon the rate of transbilayer lipid exchange (flip-flop) of PC molecules from the inner to the outer monolayer. The vesicles only become permeable to the Dy3+ ions when lysoPC is mobilised in the flip-flop process of exchange of lipid molecules between the two monolayers. The n-alcohols affect both the time taken to initiate flip-flop of inner monolayer PC and the subsequent rate of permeability to Dy3+. The n-alcohols are seen to affect all the above rates in an identical chain-length dependent manner, indicating a common cause for all observations which we identify as the degree of clustering of the n-alcohol molecules in the bilayer. The results are discussed in terms of the chain-length dependent mechanism of n-alcohol interactions with the membrane and the mechanism by which the vesicles become permeable to Dy3+ ions.