Molecular weight estimation of protoheme ferro-lyase by radiation inactivation.

The molecular weight of protoheme ferro-lyase [EC 4.99.1.1], a mitochondrial enzyme, was estimated by the radiation inactivation method. Irradiation in vacuo caused less inactivation than that in air. The approximate molecular weight determined by this method was in the range from 250,000 to 320,000. The significance of the location of the enzyme in connection with its role in heme synthesis is discussed on the basis of Singer's membrane theory.     

Relation between electrophoretic esterase patterns and organophosphate resistance in Musca domestica

An Italian OP-resistant strain C turned out to be heterogeneous for a gene on the 5th chromosome causing a difference in the mobility of an esterase in electrophoresis. Individuals containing only band 1 or 2 are homozygous for either allele, individuals containing esterase 1 and 2 are heterozygous. Two substrains were derived, E₁ and E₂, homozygous for the allele for 1 and 2 respectively. These strains were found to be remarkably different in OP-resistance. E₁ is approximately equally resistant as strain C, and contains the breakdown enzyme degrading paraoxon and diazoxon. E₂ has a lower resistance and does not contain the breakdown enzyme. The presence of the breakdown enzyme and the 1 esterase are controlled by the same gene allele, but they are certainly not identical. For instance the 1 band is quite stable, and is in solution in normal homogenates, whereas the breakdown enzyme is very labile and particulate. Therefore one allele, a _C1, controls both band 1 and the breakdown enzyme, the other, a _C2, seems to control only band 2. A similar situation was found in a susceptible strain, bwb; ocra, which had previously been shown to be heterogeneous for the ali-esterase a. Two alleles of the a gene are present here: the a ⁺allele controls the presence of band 1 and ali-esterase a; the a ₂allele seems to control band 2 only. The electrophoretic speed of the breakdown enzyme was found to be approximately equal to that of esterase 2.

---

Zusammenfassung Ein italienischer OP-resistenter Musca domestica-Stamm erwies sich als heterogen für ein Gen im 5. Chromosom, das einen Unterschied in der Beweglichkeit einer Esterase bei der Elektrophorese bedingt. Individuen, die nur die Bande 1 oder 2 enthalten, sind homozygot für jeweils das eine der beiden Allele. Individuen, welche die Esterase 1 und 2 enthalten, sind heterozygot. Es wurden zwei Unterstämme E₁ und E₂ abgezweigt, die jeweils für das Allel 1 und 2 homozygot sind. Diese Stämme erwiesen sich als bemerkenswert verschieden hinsichtlich der OP-Resistenz. E₁ ist annähernd so resistent wie Stamm C und enthält das Abbau-Enzym, welches Paraoxon und Diazoxon zerstört. E₂ hat eine geringere Resistenz und enthält kein Abbau-Enzym. Die Gegenwart des Abbau-Enzyms und der 1-Esterase werden von dem gleichen Genallel kontrolliert, sind aber sicher nicht identisch. Z.B. ist Bande 1 ganz stabil und in normalen Homogenisaten in Lösung, während das Abbau-Enzym sehr labil und ungelöst ist. Deshalb kontrolliert das eine Allel, a _C1, sowohl Bande 1 und das Abbau-Enzym, das andere, a _C2, anscheinend nur Bande 2. Eine ähnliche Situation wurde in dem anfälligen Stamm, bwb; ocra, gefunden, der sich bereits früher als heterozygot für die Ali-Esterase a erwiesen hatte. Dabei sind 2 Allele des a-Gens vorhanden: das a ⁺-Allel kontrolliet die Anwesenheit von Bande 1 und Ali-Esterase a; das a ₂-Allel scheint nur Bande 2 zu kontrollieren. Die Elektrophorese-Geschwindigkeit des Abbau-Enzyms erwies sich als annähernd so groß wie die der Esterase 2.

---

---

When the first author died in October 1964, he left the material for this publication. It is a tribute to his accuracy that it was possible for the last author to prepare this paper from his notes.     

Effects of DNA charge and length on the electrophoretic mobility of intercalated DNA

DNA molecules ranging in size from 1 to 630 kilobase pair and intercalated with either ethidium bromide (EtBr) or propidium iodide (PI) were electrophoresed in 1% agarose at four different electric field strengths. The extent of intercalation of EtBr under the conditions of our electrophoresis experiments was determined by a spectroscopic technique, whereas the extent of intercalation of PI was inferred from previous studies. The effects of the increase in DNA contour length and the concomitant decrease of linear charge density were separated based on our analysis of the mobility data. We conclude that the main factor responsible for the reduced electrophoretic mobility of intercalated DNA is the diminished linear charge density and not the increased contour length. © 1994 John Wiley & Sons, Inc.     

Platelet activating factor activity in the phospholipids of bovine spermatozoa

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of [3H]serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.     

Ionic charge on phospholipids and their interaction with the mitochondrial adenosine triphosphatase

The activity of the lipid-depleted, oligomycin-sensitive mitochondrial ATPase has been measured in the presence of liposomes prepared from mixtures of phosphatidylglycerol and phosphatidylglycerol lysine. Enzyme activity increased linearly with an increase in the negative charge of liposomes prepared from the phosphatidylglycerol-phosphatidylglycerol lysine mixtures. The electrophoretic mobility and activating capacity of liposomes of several other phospholipids were determined. A linear relationship between electrophoretic mobility of the liposomes and oligomycin-sensitive activity was again apparent. These observations demonstrate that the activity of the ATPase is directly proportional to the ionic charge on phospholipid activators if the acyl chain composition of the phosphoglycerides is relatively constant.     

The influence of charge and the distribution of charge in the polar region of phospholipids on the activity of UDP-glucuronosyltransferase.

Studies of the mechanism of lipid-induced regulation of the microsomal enzyme UDP-glucuronosyltransferase have been extended by examining the influence of charge within the polar region on the ability of lipids to activate delipidated pure enzyme. The effects of net negative charge, of charge separation in phosphocholine, and of the distribution of charge in the polar region of lipids were studied using the GT2p isoform isolated from pig liver. Prior experiments have shown that lipids with net negative charge inhibit the enzyme (Zakim, D., Cantor, M., and Eibl, H. (1988) J. Biol. Chem. 263, 5164-5169). The current experiments show that the extent of inhibition on a molar basis increases as the net negative charge increases from -1 to -2. The inhibitory effect of negatively charged lipids is on the functional state of the enzyme and is not due to electrostatic repulsion of negatively charged substrates of the enzyme. Although the inhibitory effect of net negative charge is removed when negative charge is balanced by a positive charge due to a quaternary nitrogen, neutrality of the polar region is not a sufficient condition for activation of the enzyme. In addition to a balance of charge between Pi and the quaternary nitrogen, the distance between the negative and positive charges and the orientation of the dipole created by them are critical for activation of GT2p. The negative and positive charges must be separated by the equivalent of three -CH2- groups for optimal activation by a lipid. Shortening this distance by one -CH2- unit leads to a lipid that is ineffective in activating the enzyme. Reversal of the orientation of the dipole in which the negative charge is on the polymethylene side of the lipid-water interface and the positive charge extends into water also produces a lipid that is not effective for activating GT2p. On the other hand, lipids with phosphoserine as the polar region, which has the "normal" P-N distance but carries a net negative charge, do not inhibit GT2p. This result again illustrates the importance of the dipole of phosphocholine for modulating the functional state of GT2p.     

Ionic charge on phospholipids and their interaction with the mitochondrial adenosine triphosphatase.

The activity of the lipid-depleted, oligomycin-sensitive mitochondrial ATPase has been measured in the presence of liposomes prepared from mixtures of phosphatidylglycerol and phosphatidylglycerol lysine. Enzyme activity increased linearly with an increase in the negative charge of liposomes prepared from the phosphatidylglycerol-phosphatidylglycerol lysine mixtures. The electrophoretic mobility and activating capacity of liposomes of several other phospholipids were determined. A linear relationship between electrophoretic mobility of the liposomes and oligomycin-sensitive activity was again apparent. These observations demonstrate that the activity of the ATPase is directly proportional to the ionic charge on phospholipid activators if the acyl chain composition of the phosphoglycerides is relatively constant.     

Investigation on the interactions between pirarubicin and phospholipids

Differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy and quantum calculation based on molecular modeling were applied to investigate the interaction between pirarubicin (THP), an anthracycline antibiotic frequently used in chemotherapy, and zwitterionic distearoylphosphatidylcholine (DSPC) or anionic distearoylphosphatidylglycerol (DSPG). DSC and FTIR studies suggested that DSPG bilayers were less perturbed by THP than those of DSPC, and this might be due to the strong interactions between NH₃⁺ of THP and the phosphate (PO₂⁻) group in the polar head of DSPG, which limit the further access of THP into its bilayers. Quantum calculation results based on molecular modeling could further confirm the DSC and FTIR conclusions. Meanwhile, it could well translate the calorimetric and spectroscopic phenomena into the underlying physical knowledge. Interactions between THP and phospholipids can play a critical role in the liposomal drug delivery system, especially in the safety mechanism elucidation and rational formulation design.     

Inhibition of protohaem ferro-lyase by N-substituted porphyrins. Structural requirements for the inhibitory effect.

N-Methyl mesoporphyrin was a powerful inhibitor of protohaem ferro-lyase in vitro, whereas N-ethyl mesoporphyrin and N-methyl coproporphyrin were not and neither was the newly described green pigment produced by giving rats ethylene. This suggests that the size of the substituent at a pyrrole nitrogen and also the number of carboxylic acid side chains of the substituted porphyrin are important for the inhibitory effect. Evidence that N-methyl mesoporphyrin inhibited the enzyme, whereas the ethylene-derived pigment did not, was also obtained in vivo.     

Surface charge of endothelial cells estimated from electrophoretic mobility

The surface charge density of endothelial cells was estimated from cell electrophoresis. Cultured endothelial cells from the bovine pulmonary artery were suspended in saline and placed in the lumen of a glass capillary. A voltage was applied across the capillary ends and the velocity imparted to the cells was measured with a microscope. Erythrocyte mobility was also measured. The mobility in (micron/s)/(V/cm) was 0.74 +/- 0.08 for endothelial cells and 1.03 +/- 0.15 for erythrocytes. Charge density in esu/cm2 was calculated as 2.62 x 10(4) and 0.91 x 10(4) for endothelial and red cells, respectively. Removal of sialic acid did not affect the mobility of endothelial cells, but it reduced that of red cells to near zero. Endothelial cell mobility decreased either with ionic strength or calcium concentration. Our results strongly suggest that the surface charge of endothelial cells is dependent on sulfated glycosaminoglycans.     

Molecular charge and electrophoretic mobility in cetacean myoglobins of known sequence

Fourteen myoglobins of known sequence were examined by polyacrylamide gel electrophoresis at five pH values. Gels at each pH divided the sequences into six to eight distinct classes, while the combination of the results of three gels at different pH levels distinguished 13 of 14, or 93%, of the sequences. The relative mobility of the myoglobins in the gels is significantly correlated with the charges of the proteins calculated from the pK values of the ionized groups. Major differences in mobility corresponded to expected differences in charge due to the amino acid substitutions between sequences. In addition to sequences differing in the total number of acidic and basic residues, those differing from each other in the total number of histidines were distinguished on low-pH gels. One pair of sequences differing by the exchange of lysine for arginine was separated on high-pH gels, as predicted from the differences in ionization of these two amino acids. On gels at pH 10.4, there was greater deviation of electrophoretic mobility from charge than on other gels, possibly due to the influence of amino acid substitutions in the neighborhood of lysine residues. Manipulation of the concentration and composition of the gels did not change the separation of the sequences from each other. Examination of myoglobins by gel electrophoresis at a wide range of pH values allows discrimination of nearly all amino acid substitutions and demonstrates the close relationship between titration and relative electrophoretic mobility.This work was supported by NIH Grants GM 24849 to R. C. Lewontin and CA 28854 to M. Skolnick.     

Relation between the structure of alliin analogues and their inhibitory effect on platelet aggregation

Alliin analogues have been synthesized and tested for their inhibitory activity on platelet aggregation. It is found that only allicin, the S-oxodiallyl disulphide, has a strong inhibitory effect, comparable to that of alliin, while all the other tested compounds do not show any inhibitory effect even at concentrations of 10^?3 M.