A test of the human sperm acrosome reaction following ionophore challenge. Relationship to fertility and other seminal parameters.

Acrosome reaction capacity was tested on semen samples from 53 fertile and 26 subfertile men. Preparations were divided into two aliquots after 3 or 24 hours of culture. One aliquot received 10 mumol/L calcium ionophore A23187 in dimethyl sulfoxide (DMSO) and the other received DMSO alone. Acrosome reactions were scored on ethanol-permeabilized smears using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum lectin. The following factors were analyzed: the spontaneous reaction rates (control); induced reaction rates (ionophore-challenged); and the difference between the two, being the proportion of spermatozoa in the population capable of reacting in response to calcium influx (acrosome reaction to ionophore challenge [ARIC]). While spontaneous reactions bore no relation to fertility, induced reactions and ARICs were significantly reduced or absent in subfertile men, indicating acrosomal dysfunction as a likely cause of fertilization failure. The test was shown to have a predictive value for fertility comparable to that of the hamster ovum sperm penetration assay and to be a simple and cost-effective addition to existing semenology.

Differences in myeloperoxidase activity from neutrophilic polymorphonuclear leukocytes of differing density: relationship to selective exocytosis of distinct forms of the enzyme.

Elicited murine neutrophilic polymorphonuclear leukocytes (PMN) were fractionated by Percoll density gradient centrifugation into high density (HD) and intermediate density (ID) populations. As described in the accompanying article HD- and ID-PMN appear to represent "resting" and "activated" cell populations, respectively. Consistent with this possibility, histochemical and biochemical evidence suggested that ID-PMN were degranulated compared to HD-PMN. Myeloperoxidase (MPO) in the ID-PMN population showed increased sensitivity to inhibition by 3-amino-1,2,4-triazole, and HD-PMN exhibited a 2-3-fold increase in chloride and iodide oxidation per unit of MPO activity compared to ID-PMN. When HD-PMN were induced to degranulate in vitro, the remaining cell-associated MPO displayed enzymatic properties characteristic of the activity associated with ID-PMN. The mechanism of this phenomenon was also investigated in vitro using purified human peripheral blood PMN and the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. Differences in cell-associated MPO activity were shown to be related to selective exocytosis of enzymatically and chromatographically distinct forms of the enzyme. These data indicate that, in addition to the well known selective exocytosis of specific and azurophilic granules induced by various agents, selectivity may also occur at the level of enzymatically distinct forms of a particular granule enzyme. Moreover, our observations provide further evidence that density differences may be utilized to fractionate and study the generation of functionally distinct subpopulations of PMN that arise in vivo as well as in vitro following exposure to various stimuli.

Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxygenase. Kinetics of the reductive activation of the enzyme.

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.

Multiple forms of myeloperoxidase from human neutrophilic granulocytes: evidence for differences in compartmentalization, enzymatic activity, and subunit structure.

Multiple forms of myeloperoxidase from normal human neutrophilic granulocytes obtained from a single donor can be resolved by carboxymethyl (CM)-cellulose ion-exchange column chromatography into three forms (I, II, and III) designated in order of elution of adsorbed enzyme using a linear salt gradient. Selective solubilization of individual forms of the enzyme by detergent (form I) or high-ionic-strength procedures (forms II and III) suggested that these forms of the enzyme were compartmentalized differently. All three forms were purified by a combination of preferential extraction, manipulation of ionic strength, and ion-exchange and molecular sieve chromatography. Purified forms II and III had similar specific activities for a variety of substrates. Form I was less active toward several of these same substrates, most notably iodide, with a specific activity about one-half that of forms II and III. All forms had similar spectral properties characteristic of a type alpha heme. The amino acid compositions of the three forms were similar, yet significant differences were found in selected residues such as the charged amino acids. Native polyacrylamide gel electrophoresis resolved small differences in mobility between the forms which were consistent with the charge heterogeneity observed on CM-cellulose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis data were consistent with the generally accepted subunit structure of two heavy chains and two light chains. All three forms contained a small-molecular-weight subunit of Mr 11,500. Form I contained a large subunit of Mr 63,000, while forms II and III contained a corresponding subunit of Mr approximately 57,500. We conclude that heterogeneity of human myeloperoxidase is accompanied by differences in cellular compartmentalization, enzymatic activity, and subunit structure.

Density heterogeneity of neutrophilic polymorphonuclear leukocytes: gradient fractionation and relationship to chemotactic stimulation.

When elicited murine peritoneal exudate cells were subjected to Percoll density gradient centrifugation, polymorphonuclear neutrophils (PMN) were found to distribute over a broad spectrum of buoyant densities (1.10-1.06 g/ml). PMN isolated between approximately 1.10 and 1.085 g/ml were referred to as high density PMN (HD-PMN), and those isolated at approximately 1.085-1.06 g/ml were designated intermediate density PMN (ID-PMN). Cells were characterized on the basis of morphology and specific markers: PMN by lactoferrin immunocytofluorescence and macrophages by nicotinamide adenine dinucleotide glycohydrase activity. Macrophages banded near the top of the gradient with a peak at 1.04 g/ml. At increasing times following elicitation, the ratio of HD to ID-PMN decreased. Decreased density of either murine HD-PMN or human peripheral blood PMN could be induced in vitro by exposure of the cells to endotoxin-activated serum. A decrease in buoyant density of human PMN was also demonstrated in vitro using the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The response was time dependent, related to dose, and appeared to be mediated by the cell membrane receptor for FMLP. A competitive antagonist of FMLP binding, carbobenzoxy-phenylalanyl-methionine, inhibited the density change with a calculated Kd similar to that reported for inhibition of FMLP-induced aggregation, degranulation, locomotion, and superoxide production. The FMLP-induced decrease in PMN density was shown to be directly correlated with increases in relative mean cell volume. The density response is a new measurement of PMN interaction with specific chemotactic factors, which may be important in the generation of PMN heterogeneity observed in elicited peritoneal exudate cells. In addition, this approach offers a means of physically separating "activated" from "resting" PMN and of studying resultant biochemical differences between these cell populations using both in vivo and in vitro systems.

Mechanistic studies on phenylalanine hydroxylase from Chromobacterium violaceum. Evidence for the formation of an enzyme-oxygen complex.

Steady-state kinetic analysis of pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum indicated that the enzyme follows a partially ordered reaction mechanism. The data suggested that oxygen is the first substrate to bind to the enzyme. This result was further supported by rapid-quench experiments in which the enzyme-oxygen complex was trapped to yield product. Additional support for the presence of an enzyme-oxygen complex was derived from magnetic susceptibility measurements of molecular oxygen in the presence and absence of cuprous phenylalanine hydroxylase. The magnetic susceptibility of dissolved oxygen decreased in the presence of the enzyme, supporting a direct oxygen-metal interaction.

Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome.

A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.

Differential distribution of distinct forms of myeloperoxidase in different azurophilic granule subpopulations from human neutrophils.

Myeloperoxidase (MPO), a characteristic enzyme of human polymorphonuclear neutrophils (PMN), is localized in specialized lysosomal or azurophilic granules, and can be resolved into three distinct forms (I, II, III) by ion-exchange chromatography. Granules were isolated from single donor PMN and fractionated with centrifugation into two different azurophilic subpopulations (high and low density) by banding in a continuous sucrose density gradient. Ion-exchange chromatography of granule extracts indicated that the lower density granules contained mainly MPO forms II and III while the higher density granules appeared to contain all three forms, but in much reduced amounts. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed that, the mobilities of the heavy subunits of MPO appeared to be inversely related to the density of the granule population from which they were extracted. These observations suggest that the different forms of MPO may have distinct functional roles and/or are a possible reflection of maturational differences among the granule subpopulations.

Adduct formation between the cupric site of phenylalanine hydroxylase from Chromobacterium violaceum and 6,7-dimethyltetrahydropterin.

The interaction of pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum with the cofactor analogue 5-deaza-6-methyltetrahydropterin and the cofactor 6,7-dimethyltetrahydropterin (DMPH4) has been investigated by multifrequency electron spin resonance (ESR) spectroscopy. 5-Deaza-6-methyltetrahydropterin, which lacks the N-5 nitrogen present in the pyrazine ring of DMPH4, binds tightly to the cupric form of the enzyme; however, no changes are observed in the ESR parameters of the copper center. In contrast, the binding of DMPH4 (or 6-methyltetrahydropterin) shifts the ESR parameters (g and A) associated with the cupric enzyme. In addition, superhyperfine transitions were resolved and assigned to hyperfine splitting from nitrogen ligands. ESR spectra of the enzyme recorded in the presence of [5-14N]DMPH4 or [5-15N]DMPH4 were computer simulated and found to be consistent with pterin serving as a direct donor ligand to the copper center through the N-5 position.