Quantitative evidence of a silent gene P° of human red cell acid phosphatase in south Polish population

The distribution of red cell acid phosphatase types in 3244 unrelated Polish adults is reported. Gene frequencies P^a = 0.3594, P^b = 0.5643 and P^c = 0.0763 were obtained. In a forensic case of disputed paternity an apparent mother/child incompatibility respect to red cell acid phosphatase was found, the mother appearing as type B and the child as type A. Determination of acid phosphatase activity suggested the presence of a silent gene P°. The phosphatase levels were about half the values expected, as determined in 237 adults representing the different phenotypes.     

Zur Populationsgenetik der sauren Phosphatase der Erythrocyten (E C 3.1.3.2): Phänotypen-und Allelhäufigkeiten in Südwestdeutschland

Summary Within an population sample of 300 individuals of Southwestern Germany the red cell acid phosphatase polymorphism is investigated. Gene frequency estimates are: P^a=0.31, P^b=0.643, P^c=0.047.

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Direktor: Prof. Dr. med. Dr. H. Baitsch

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Red cell isozymes in the Eastern Carolines

Four populations of islanders (Ponapeans, Mokilese, Pingelapese, and Kusaieans) in the Eastern Caroline Islands have been surveyed for variability in red cell acid phosphatase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, adenylate kinase and glucose-6-phosphate dehydrogenase. The following gene frequencies were observed: P^a = 0.0904, PGM²₁ = 0.1015, and PGD^B = 0.0259. No genetic variation was encountered in the AK and G6PD systems.     

Human red cell esterase D polymorphism in Denmark, its use in paternity cases and the description of a new phenotype

Red cell esterase D (EsD) phenotypes were determined in a Danish population sample of 3,116 unrelated adults by starch-gel electrophoresis. A new phenotype was discovered, which appeared to be determined by the EsD1 allele and a new allele EsDCph. The gene frequencies observed were EsD1 = 0.9007, EsD2 = 0.0992, EsDCph = 0.0001. Investigation of 1,111 mother-child pairs and 59 families with 157 offspring added further support to the genetic model of two common alleles at an autosomal locus. The applicability of the EsD polymorphism to paternity testing was investigated on 960 cases of disputed paternity. An estimate of the EsD null allele frequency (0.001) in European populations was made on the basis of observations made on 5,864 mother/child combinations and 762 matings with 1,882 offspring. The influence of this allele on the reliability of exclusions of paternity was determined.     

On the phenotype distribution of red cell acid phosphatase in Czechoslovakia

Summary A random group of 300 donors living in the district of eské Budjovice (southern Bohemia) have been investigated for the incidence of the individual phenotypes of red cell acid phosphatase. The calculated frequencies of the genes P^a, P^b and P^c are 0.322, 0.615 and 0.063, respectively.     

Pseudocholinesterases and human red cell acid phosphatases in Koreans

Summary The authors reveal the results of pseudocholinesterase and human red cell acid phosphatase typings in a sample of 115 unrelated female Koreans aged from 20–30. No atypical pseudocholinesterase variants could be demonstrated. The frequencies of human red cell acid phosphatase alleles run up to: ph^A=0.231, ph^B=0.769, ph^C=0.000.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Zur Populationsgenetik der sauren Phosphatase der Erythrocyten (EC: 3.1.3.1): Phänotypen- und Allelhäufigkeiten in der ČSSR

Summary Within a population sample of 307 blood donors of Prague the polymorphism of the red cell acid phosphatase has been investigated. Gene frequency estimates are: P^a=0.365, P^b=0.578, P^c=0.057.     

The rare silent gene Po of human red cell acid phosphatase in a second family in poland

The authors describe a Polish family (three generations) with a silent gene P^o of human red cell acid phosphatase. The phosphatase levels of 4 family members were about half the normal value.     

Occurrence of the ACP 1 0 allele in Czechoslovakia

Summary During a paternity test an unexpected type of red cell acid phosphatase isozyme (ACP₁) was found in one family. The mother was of type A and type B was diagnosed in the son. The whole family was then subjected to ACP₁ phenotyping and to the enzyme assay. Five members of the family were found to have unexpected types of ACP₁ isozymes. The average activity was approx. 50% of normal values. It is presumed that a silent ACP ₁ ⁰ allele was found in the family investigated and that the grandfather was its first carrier.     

All or none cell responses of Ca2+-dependent K channels elicited by calcium or lead in human red cells can be explained by heterogeneity of agonist distribution

Summary We have studied the all or none cell response of Ca²⁺-dependent K⁺ channels to added Ca in human red cells depleted of ATP by incubation with iodoacetate and inosine. A procedure was used which allows separation and differential analysis of responding and nonresponding cells. Responding (H for heavy) cells incubated in medium containing 5mM K lose KCl and water and increase their density to the point of sinking on diethylphthalate (specific gravity=1.12) on centrifugation. Nonresponding (L for light) cells do not lose KCl at all. There is no intermediate behavior. Increasing the Ca concentration in the medium increases the fraction of cells which become H. No differences in the sensitivity to Ca²⁺ of the individual K⁺ channels were detected in inside-out vesicles prepared either from H or from L cells. The Ca content of H cells was higher than that of L cells. Cells depleted of ATP by incubation with iodoacetate and inosine sustain pump-leak Ca fluxes of about 15 mol/liter cells per hour. ATP seems to be resynthesized in these cells at the expense of cell 2,3-diphosphoglycerate stores at a rate of about 150 mol/liter cells per hour. Inhibition of 2,3-diphosphoglycerate phosphatase by tetrathionate increased 6–8 times the measured rate of uptake of external⁴⁵Ca. This was accompanied by an increase in the fraction of H cells. All or none cell responses of Ca²⁺-dependent K channels have also been evidenced in intact human red cells on addition of Pb. They have the same characteristics as those in responding and nonresponding cells. The detailed study of the kinetics of Pb-induced shrinkage of red cells suspended in medium containing 5mM K showed that changes of Pb concentration changed not only the fraction of H cells but also the rate of shrinkage of responding cells. H cells generated by Pb treatment contained significantly more lead than L cells. The above results suggest that the two all or none cell responses studied here can be explained by heterogeneity of agonist distribution among cells. Since pump-leak fluxes exist in both cases, differences of agonist distribution could be generated by heterogeneity of pumping among cells. This interpretation turns interest from K channels to Ca pumps to explain the heterogeneous behavior of red cells in response to a uniform stimulus.     

Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina

A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,l-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm^-3. Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm^-3. Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - RuBP carboxylase D-ribulose-1,5-bisphosphate carboxylase or 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39     

Ruthenium red selectively depletes inositol 1,4,5-trisphosphate-sensitive calcium stores in permeabilized rabbit pancreatic acinar cells

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca²⁺/mg protein in an energy-dependent pool when incubated at an ambient free Ca²⁺ concentration of 100 nm. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca²⁺ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca²⁺ efflux and ATP-driven Ca²⁺ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P₃, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 m, selectively reduced the size of the Ins(1,4,5)P₃-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P₃. At concentrations beyond 100 m, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca²⁺ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca²⁺ from the Ins(1,4,5)P₃-sensitive store by activating a Ca²⁺ release channel, whereas Ca²⁺ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca²⁺ pump. The postulated ruthenium red-sensitive Ca²⁺ release channel might be similar to the ryanodine-receptor in muscle.The research of Dr. P.H.G.M. Willems has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.     

Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K _Ca ) channel and (iii) a small K⁺ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na⁺ and K⁺, but not permeable to Cl⁻ or N-methyl-d-glucamine. Channel activity required the presence of Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ concentrations as low as 10⁻⁷ m (open probability (P _o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P _o of the NSC channel from both the external and cytosolic side (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K⁺ could be fitted to the I-V relationship in asymmetrical K⁺ and Na⁺ solutions. The channel was impermeable to Cl⁻ and N-methyl-d-glucamine. P _o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P _o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca²⁺ activated, voltage-insensitive and involved in both constitutive K⁺ and Na⁺ reabsorption from endolymph while the BK channel might participate in the K⁺ pathway under stimulated conditions that produce an elevated intracellular Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

Different arachidonate and palmitate binding capacities of the human red cell membrane

Human red cell membrane bindings of arachidonate and palmitate at pH 7.3 are investigated at temperatures between 0 and 38°C by equilibrating ghosts with the long-chain fatty acids bound to bovine serum albumin in molar ratios (v) within the physiological range (<1.7). Linearized relations of ghost uptakes and fatty acid monomer concentrations in buffer provide estimates of the binding capacities and corresponding equilibrium dissociation constants (K _dm ). The temperature-independent arachidonate binding capacity, 5.5 ± 0.5 nmol g^–1 packed ghosts, is approximately fivefold smaller than that of palmitate, 26.6 ± 2.0 nmol g^–1. While K _dm of arachidonate binding 5.1 ± 0.5 nm is temperature independent, K _dm of palmitate increases with temperature from 3.7 nm at 0°C to 12.7 nm at 38°C.The large difference in binding capacities suggests the presence of at least two different fatty acid binding domains in human red cell membranes.     

Population,formal genetics,and linkage relations of the phosphoglycolate phosphatase (PGP)—E.C.3.1.3.18

Summary One hundred and sixty-seven blood donors, 26 families with 72 offspring and 12 motherchild couples were studied for the phosphoglycolate phosphatase polymorphism. In hemolysates, the isozymes are stable for at least five weeks. The distribution of observed phenotypes in the population study did pot diverge from the expected values according to Hardy-Weinberg law. In the family study, the formal genetic model of three alleles—PGP ¹, PGP ² and PGP ³ at one autosomal locus-could be confirmed. Among 33 individuals from a Mongoloid population PGP 1 was observed in 100%. This observation lead us to the conclusion, based also on recent data in Negroid populations (Barker and Hopkinson 1978), that phosphoglycolate phosphatase may be a more recent polymorphism of Caucasoid populations. Linkage studies with the hp locus an chromosome 16 resulted in 19 meiotic divisions of 4 informative families in a lod score peak of 0.23 at =0.25 being inconclusive. The inclusion of the PGP system in paternity testing is also discussed.     

Some notes on the geographical distribution of the human red cell acid phosphatase phenotypes

Summary Basing on the data of 65 populations the geographical variability of the human red cell acid phosphatase phenotypes resp. alleles was studied. We found a marked distribution gradient: The frequency of p^B-alleles increases with the increase of the mean annual temperature of the various biotops, whereas the p^A-allele frequencies show a clear decrease. For this allele we calculated a significant negative correlation between its frequency and the mean annual temperature: r=-0.71; P<0.001. We suppose that the p^B-allele is in some way adaptive under the climatic conditions of tropical biotops. The possible reasons are discussed.

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Zusammenfassung An Hand der Angaben von 65 Populationen wurde die geographische Variabilität in der Phänotypen- und Allelenverteilung der menschlichen sauren Erythrocytenphosphatasen untersucht. Dabei ergab sich ein deutlicher Verteilungsgradient insofern, als die p^B-Frequenz mit zunehmender mittlerer Jahrestemperatur zunimmt, während die p^A-Frequenz abnimmt. Für dieses Allel konnte eine signifikante negative Korrelation zwischen seiner Frequenz und der mittleren Jahrestemperatur ermittelt werden: r=-0,71; P<0.001. Wir vermuten, daß das p^B-Allel unter den klimatischen Bedingungen tropischer Biotope adaptiver ist. Die möglichen Ursachen hierfür werden diskutiert.

     

Testing the Hypothesis that System y<Superscript>+</Superscript>L Accounts for High- and Low-Transport Phenotypes in Chicken Erythrocytes Using <Emphasis Type="SmallCaps">L</Emphasis>-Leucine as Substrate

Experiments were carried out to test the hypothesis that system y⁺L accounts for the high (HT) and low (LT) amino-acid transport phenotypes in chicken erythrocytes and to explain the different effect of selective breeding on lysine and leucine fluxes. L-Leucine transport was characterized in individuals which had been separated into two groups (HT and LT) according to their capacity to transport L-lysine across the erythrocyte membrane. Whereas lysine influx (1 μM) in the two groups differed by 32-fold (HT/LT), leucine influx was not significantly different. Average rates (nmol/ L cells/ min) were: 227 (HT) and 7.0 (LT) for L-lysine, and 8.9 (HT) and 7.1 (LT) for L-leucine. The differential ability of L-lysine and L-leucine fluxes to discriminate between the HT and LT phenotypes was shown to be consistent with the interactions of these substrates with system y⁺L and to vary depending on the conditions of the assay. It is shown that the two phenotypes can be clearly discriminated by measuring L-leucine influx in the presence of Li⁺. These results support the hypothesis that the HT and LT phenotypes reflect alterations in the function of system y⁺L and illustrate that the choice of the appropriate substrate and medium composition must be carefully considered when investigating the consequences of either experimental or natural alterations of broad-scope transporters.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Polymorphism of the red cell acid phosphatase in the Swiss population

Summary The distribution of the red cell acid phosphatase groups was studied on 1365 blood samples of Swiss individuals. The distribution is in agreement with the Hardy-Weinberg equilibrium. Gene frequencies similar to those observed in other Caucasian populations were obtained (P^A=0.345, P^B=0.607, P^C=0.049). In 331 mother/child-combinations, we found no theoretically impossible combinations.     

The purification and kinetic properties of biophosphoglycerate synthase from horse red blood cells.

Bisphosphoglycerate synthase from horse red cells has been purified to apparent homogeneity by a simple and efficient new procedure incorporating chromatography on a column of Sepharose 4B derivatized with blue dextran. The enzyme is similar to the human red cell synthase in subunit size. It is phosphorylated by either glycerate-1,3-P₂ or glycerate-2,3-P₂ to form a phosphoenzyme with the acid-lability of a histidyl phosphate. In addition to the synthase activity (glycerate-1,3-P₂ → glycerate-2,3-P₂), k_cat 12.5 s^?1, the enzyme has bisphosphoglycerate phosphatase activity in the presence of glycolate-2-P (glycerate-2,3-P₂ → glycerate-P + P_i), k_cat 2.6 s^?1 and phosphoglycerate mutase activity (3-PGA ? 2-PGA), k_cat 1.7 s^?1. The energy of activation for the synthase reaction is 9.38 kcal/mol. Lineweaver-Burk plots of the kinetic data are parallel lines. In contrast intersecting patterns were obtained from similar experiments done with the human red cell enzyme. Further investigation is required to explain these differences. This enzyme may function as both synthase and phosphatase for bisphosphoglycerate in the red blood cell.