The pH dependence of the cardiac sarcolemmal Ca2(+)-transporting ATPase: evidence that the Ca2+ translocator bears a doubly negative charge

The pH dependence of the Ca2(+)-transporting ATPase of bovine cardiac sarcolemma was determined in a membrane vesicle preparation. The maximal velocity (Vmax) at saturating external Ca2+ showed a sigmoidal pH dependence with maximal values in the 6.0-6.5 range, a half-maximal value at 7.2 and minimal (less than or equal to 15%) values at pH greater than or equal to 8.0. The apparent affinity for Ca2+ (1/Km) varied over 10(4)-fold for 6.0 less than or equal to pH less than or equal to 8.5, increasing with increasing pH. Plots of log(1/Km) vs. pH were biphasic. In the acid range (6.0 less than or equal to pH less than or equal to 7.2), a slope of 2.6 was observed for the calmodulin-activated form of the pump. For 7.2 less than or equal to pH less than or equal to 8.5, a slope of 0.5 was observed. At pH 7.4, the Km is approx. 48 +/- 19 nM. The Ca2+ pump of cardiac sarcoplasmic reticulum in the same preparation had a Km of 304 +/- 115 nM and showed a similar pH dependence except that the slope in the acid range was 1.7. When calmodulin was removed from the sarcolemmal pump, its Km was raised to approx. 1.0 microM, the slope in the acid range was reduced to 1.7 and the Vmax was markedly reduced. The results are explicable in terms of a model in which each of the two Ca2+ binding sites on the pump contains two buried COO- groups responsible for high affinity. The Km effect is explained by 2 H+ vs. 1 Ca2+ competition for occupation of each of the two cytoplasmically-oriented translocators (4 H+ vs. 2 Ca2+). The Vmax effect is explained by counter-transport of H+. The findings are considered in terms of the published amino acid sequence of the cardiac sarcolemmal pump and recent site-directed mutagenesis vs. function studies identifying the Ca2+ binding site in the skeletal sarcoplasmic reticulum pump. The kinetic data are also applied to pump behavior under conditions of ischemia and acidosis.     

Slow inactivation and reactivation of the K+ channel in squid axons. A tail current analysis.

Potassium current inactivation and reactivation in squid axons were measured from tail current amplitudes after voltage clamp prepulses to the potassium equilibrium potential, EK, in seawater containing elevated levels of potassium ion concentration, Ko. Little or no inactivation resulted with prepulses lasting less than 100 ms. Longer pulses caused the current to inactivate in two phases, one between 0.1 and 1 s, and a second phase between 5 and 100 s. Inactivation was incomplete. The time constant of the tail current after a prepulse to EK was independent of pulse duration (0.1-120 s). Inactivation was independent of Ko (10 less than or equal to Ko less than or equal to 300 mM), and it was independent of membrane potential, V, for -40 less than or equal to V less than or equal to 0 mV. Reactivation was measured with a three-pulse protocol. The reactivation time course was sigmoidal with a delay of approximately 100 ms before significant reactivation occurred. These results were described by a model consisting of three inactivated states arranged in a linear sequence. The rate constants of the model are of the form (A + B exp (CV), or 1/(A + B exp (CV], which are required to describe the non-inactivating conductance component.     

Detection and identification of transient intermediates in the reactions of tryptophan synthase with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Evidence for a tetrahedral (gem-diamine) intermediate

The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy. The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively). Rate constants determined by SWSF were either well resolved (i.e., k1[DHT], k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1[OXA], k-1 greater than or equal to k2, k-2 greater than k3, k-3). The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species. Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system. The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates.     

Kinetic and magnetic resonance studies of active-site mutants of staphylococcal nuclease: factors contributing to catalysis

To determine the origin of the overall approximately 10(16)-fold rate enhancement of DNA hydrolysis catalyzed by staphylococcal nuclease, the effects of single mutations that alter the amino acid residue at each of the essential positions Asp-21, Asp-40, Thr-41, Arg-35, and Arg-87 have been examined. Metal ion and substrate analogue binding were quantitated by EPR, by the paramagnetic effects of Mn2+ on 1/T1 of water protons, and by fluorescence titrations, yielding the six dissociation constants of the ternary enzyme-Mn2+-3',5'-pdTp and enzyme-Ca2+-3',5'-pdTp complexes. The kinetic parameters kcat, KACa, KMCa, KSDNA, KMDNA, and KIMn were determined by monitoring the rate of DNA hydrolysis. By thermodynamic and kinetic criteria, Mn2+ binds tightly to the Ca2+ binding site of the enzyme but is at least 36,000-fold less effective than Ca2+ in activating the enzyme. Alterations of the liganding residues in the D40G, D40E, T41P, D21E, and D21Y mutants generally weaken the binding of Ca2+ less than or equal to 12.7-fold and of Mn2+ less than or equal to 5.4-fold, exert little effect on the KSDNA or KMDNA (less than or equal to 3.2-fold), and raise the affinity of the enzyme and its metal complexes for 3',5'-pdTp by factors less than or equal to 13.5-fold. Small changes in the ligand geometry are also reflected in the Mn2+ complexes of the liganding mutants (i.e., those in which the metal-liganding amino acids have been altered) by decreases in the electron-spin relaxation time of Mn2+. Inhibitory effects on kcat are noted in all of the liganding mutants with D40E, D40G, T41P, D21E, and D21Y showing 12-, 30-, 37-, 1500-, and greater than or equal to 29,000-fold reductions, respectively. The greater than or equal 10(3)-fold larger inhibitory effects on kcat of enlarging Asp-21 as compared to enlarging Asp-40 are ascribed to the displacement of the adjacent water molecule which attacks the phosphodiester. Mutations of each of the essential Arg residues to Gly (R35G and R87G) reduce kcat by factors greater than or equal to 35,000 but weaken metal binding less than or equal to 9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron paramagnetic resonance saturation studies of P-700+ reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T1, and spin-spin, T2, relaxation times of P-700+ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K less than or equal to T less than or equal to 100 K. T1 was 200 mus at 100 K and increased to 900 mus at 10 K. T2 was 40 ns at 40 K and increased to 100 ns at 10 K. T1 for 40 K less than or equal to T less than or equal to 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T1 deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T1 of P-700+ were examined: T1 of P-700+ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700+ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700+ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700+ due to either the iron sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700+ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of P-700+ signal. The absence of dipolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700+ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

The dimerization of ferrihaems. I. The effect of buffer ions and specific cations on deuteroferrihaem dimerization.

The dimerization of dueteroferrihaem in aqueous solution has been investigated using a parameter, named the dimerization index (Robs). This is defined as the ratio of extinction coefficients at wavelengths corresponding to Soret band maxima for the monomeric and dimeric species, respectively. For solutions containing mainly monomeric species, Robs greater than 2, whereas for solutions containing mainly dimeric species Robs less than 1. A computer programme has been applied to determine values of the dimerization constant, K, defined as: K = [dimer] [H+]/[monomer]2. Phosphate buffer anions and Tris . HCl buffer enhanced dimerization. Monovalent and divalent cations also increased dimerization, but in a specific manner. The magnitudes of their effects increased in the order K+ less than Na+ less than Li+ less than Sr2+ less than Mg2+ approximately or equal to Ca2+. Values of K were determined for several concentrations of Na+ and Sr2+. These data are interpreted in terms of a stabilization of the ferrihaem dimer by the formation of ion triplets with the added cation 'sandwiched' between carboxyl residues of the adjacent ferrihaem monomeric units. General guidelines are recommended for the choice of conditions which minimize dimerization.     

Equilibria between ionophore A23187 and divalent cations: stability of 1:1 complexes in solutions of 80% methanol/water

The cation complexation equilibria between ionophore A23187 and several alkaline earth and first transition series divalent cations have been investigated. Absorption and fluorescence spectroscopy were used to monitor the reactions which were studied in solutions of 80% methanol/water, at 25 degrees C, and under conditions of controlled ionic strength and pH. Titration of the ionophore with divalent cations results first in formation of the dimeric species MA2 and subsequently in the formation of MA+ by disproportionation of the first product. With Zn2+, Ni2+, and Co2+ (above pH approximately 6), a third species is detected which is postulated to be MA.OH. The existence of this species with Mn2+ and alkaline earth cations is uncertain. For formation of MA2, the second stepwise stability constant is similar to or exceeds the first value with all cations studied. However, it is possible to isolate the first reaction and determine accurate stability constants by working at an ionophore concentration of 3 X 10(-8) M or less and by employing pH values which preclude interference by the mixed ionophore/hydroxide species. Under these conditions, the relationship between log KMA' and pH is linear and displays a slope of 1.0. pH-independent stability constants were calculated by using pH-dependent stability constants and the known value of the ionophore's protonation constant in this solvent. The logarithms of the values obtained ranged from 7.54 +/- 0.06 for Ni2+ to 3.60 +/- 0.06 for Ba2+. The selectivity sequence and relative affinities (in parentheses) for the species MA+ are as follows: Ni2+ (977) greater than Co2+ (331) greater than Zn2+ (174) greater than Mn2+ (34) greater than Mg2+ (1.00) approximately equal to Ca2+ (0.89) greater than Sr2+ (0.20) greater than Ba2+ (0.11). Data are discussed in comparison to other studies on the complexation properties of A23187 and in terms of their significance to interpreting the transport properties of this ionophore.     

Conformation of an enzyme-bound substrate of staphylococcal nuclease as determined by NMR.

The dinucleoside phosphodiester dTdA is a slow substrate of staphylococcal nuclease (kcat = 3.8 X 10(-3) s-1) that forms binary E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. The enzyme enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of six proton resonances of dTdA, and these effects are abolished by binding of the competitive inhibitor 3',5'-pdTp. From paramagnetic effects of Co2+ on 1/T2 of phosphorus, koff of dTdA from the ternary E-Co(2+)-dTdA complex is greater than or equal to 4.8 X 10(4) s-1 and kon greater than or equal to 1.4 X 10(6) M-1 s-1, indicating the 1/T1 values to be in fast exchange. From paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with use of a correlation time of 1.6 ps on the basis of 1/T1 values at 250 and 600 MHz, 7 metal-nucleus distances and 9 lower-limit metal-nucleus distances are calculated. The long Co2+ to 31P distance of 4.1 +/- 0.9 A, which is intermediate between that expected for direct phosphoryl coordination (3.31 +/- 0.02 A) and a second sphere complex with an intervening water ligand (4.75 +/- 0.02 A), suggests either a distorted inner sphere complex or the rapid averaging of 18% inner sphere and 82% second sphere complexes and may explain the reduced catalytic activity with small dinucleotide substrates. Seventeen interproton distances and 108 lower limit interproton distances in dTdA in the ternary E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. While metal-substrate and interproton distances alone did not yield a unique structure, the combination of both sets of distances yielded a very narrow range of conformations for enzyme-bound dTdA, which was highly extended, with no base stacking, with high-anti glycosidic torsional angles for dT (64 degrees less than or equal to chi less than or equal to 73 degrees) and dA (66 degrees less than or equal to chi less than or equal to 68 degrees) and predominantly C-2'-endo sugar puckers for both nucleosides. Although the individual nucleosides are like those of B-DNA, their unstacked conformation, which is inappropriate for base pairing, as well as the conformational angles alpha and gamma of dA and zeta of dT, rule out B-DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Social selection in human populations: sufficient conditions for protection of deleterious alleles in a subdivided population

Population dynamics of wild type (A1) and the deleterious genes (A2) under social selection have been studied by considering a subdivided population where the i-th subpopulation consists of Ni individuals with relative size ci (= Ni/sigma i Ni, i = 1,2, ..., n). A social selection model is constructed by assuming that the fitness of an individual is determined by its own as well as the parental phenotypes and that the number of migrants (M) from the ith subpopulation is divided equally into other subpopulations including the ith subpopulation itself. It has been shown that the gene frequency change depends on the loss of fitness of an individual due to the trait (gamma), an affected parent in the ith subpopulation (beta i), the probability that the heterozygote develops the trait (h), and the migration rates mi (= M/Ni). For 0 less than h less than or equal to 1, a sufficient condition for protection of the deleterious allele from extinction also depends on all of these parameters. However, when mi much less than 1 for all i, the condition is beta i less than gamma/(1 - gamma) for some i, whereas when mi much greater than h[gamma + beta i(1 - gamma)] for all i it is given by sigma i ci beta i less than -gamma/(1 - gamma). When h = 0, that condition is given by sigma ici beta i less than - gamma/(1 - gamma). Analyses also show that, when the deleterious alleles in a population are rare, the relative fitnesses of A1A1, A1A2, and A2A2 are given approximately by 1, 1-hS, and 1 - S, respectively, where S is the harmonic mean of Si = gamma + beta i(1 - gamma). Thus, under mutation-selection balance, the equilibrium frequency of deleterious alleles in the entire population is given by alpha/hS for 0 less than h less than or equal to 1 and square root alpha/S for h = 0, where alpha is the irreversible mutation rate from A1 to A2 in each generation. Population dynamics of rare deleterious genes under social selection can readily be studied by considering a finite population size.     

Sodium binding sites of gramicidin A: sodium-23 nuclear magnetic resonance study.

In ethanol-water mixtures (90:10), the gramicidin dimer binds Na+ cations at well-defined sites, with a binding constant K = 4 M-1. Partial desolvation of Na+ occurs upon binding, as judged from the magnitude of the quadrupolar coupling constant (1.7 MHz) for bound sodium. The binding sites are identified with the outer sites flanking the channel entrances. The rate constants for binding and release are k+ less than or equal to 2.2 X 10(9) M-1 s-1 and k- less than or equal to 5.5 X 10(8) s-1, respectively.     

The XIST noncoding RNA functions independently of BRCA1 in X inactivation

Females with germline mutations in BRCA1 are predisposed to develop breast and ovarian cancers. A previous report indicated that BRCA1 colocalizes with and is necessary for the correct localization of XIST, a noncoding RNA that coats the inactive X chromosome (Xi) to mediate formation of facultative heterochromatin. A model emerged from this study suggesting that loss of BRCA1 in female cells could reactivate genes on the Xi through loss of the XIST RNA. However, our independent studies of BRCA1 and XIST RNA revealed little evidence to support this model. We report that BRCA1 is not enriched on XIST RNA-coated chromatin of the Xi. Neither mutation nor depletion of BRCA1 causes significant changes in XIST RNA localization or X-linked gene expression. Together, these results do not support a role for BRCA1 in promoting XIST RNA localization to the Xi or regulating XIST-dependent functions in maintaining the stability of facultative heterochromatin.     

Myofibril and sarcoplasmic reticulum changes with exercise and growth

The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), spring (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p less than or equal to 0.05). In the 51 day E group ATPase activity (0.378 +/- 0.009 mumol Pi X mg-1 X min-1) was greater than at 10 and 21 days (0.307 +/- 0.006 and 0.323 +/- 0.008 mumol Pi X mg-1 X min-1) (p less than or equal to 0.05). No change occurred in the S group from 10 to 21 and 51 days (p greater than or equal to 0.05). Both the 21 and 51 day S (0.318 +/- 0.011 and 0.399 +/- 0.010 mumol Pi X mg-1 X min-1) and E (0.323 +/- 0.008 and 0.378 +/- 0.009 mumol Pi X mg-1 X min-1) groups had higher activity compared to the C group (0.193 +/- 0.029 and 0.172 +/- 0.031 mumol Pi X mg-1 X min-1) (p less than or equal to 0.05). Maturation (10--51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca2+ binding (p less than or equal to 0.05) while Ca2+ uptake ability did not change (p greater than or equal to 0.05). SR yield, Ca2+ binding and uptake were not altered with S training (p greater than or equal to 0.05). The E training resulted in greater Ca2+ uptake at 51 days compared to C and S (p less than or equal to 0.05), with no change in Ca2+ binding (p greater than or equal to 0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.     

The structure of food webs

For nonrandom models of species interaction there is a precipitous decrease in stability as connectance increases. However, the range of stability for different models of the same connectance is large; stability also depends on how the species interactions are organized. Systems with species feeding on more than one trophic level (omnivores) are likely to be unstable, the extent depending on the number and position of the omnivores. For systems of equal connectance, those that are completely compartmentalized are less likely to be stable than those that are not.     

Quantitative evaluation of cell orientation in culture.

A quantitative method to assess mutual orientation of cells in cultures on a substrate includes the following operations: (1) the cellular groups to be evaluated are chosen; (2) position of the long axis for each nucleus of the group is determined; (3) the axis OX is arbitrary chosen for every group and the angles alphai between the long axis of every nucleus i and the axis OX are measured. Every nucleus i corresponds to a vector of unit length ei with the angles 2alpha. D, the mean of the vectors ei for every cell group is calculated. This value of D is compared with a set of values of D computed according to a model of mutual orientation studies in a simulation experiment. In this model the group of n vectors consists of a fraction of Kn parallel vectors (o less than or equal to K less than or equal to I) and of (I minus K)n randomly oriented vectors. K corresponding to the computed D which is equal to the experimental value of D is considered as an index of orientation for the group. Contact orientation with respect to the relief of the substrate may be evaluated as a root mean square deviation sigma0 to the angles between the long axes of cell nuclei and the direction of relief. Examples of the measurements of K and sigma0 in cell cultures are given.     

Complete overlap of caffeine- and K+ depolarization-sensitive intracellular calcium storage site in cultured rat arterial smooth muscle cells

Changes in the concentration of cytosolic free calcium were recorded microfluorometrically in rat vascular smooth muscle cells in primary culture and loaded with quin-2. The effects of caffeine and high extracellular K+ on the release of calcium from the intracellular storage sites were determined. In the absence of extracellular calcium, both the depolarization of plasma membrane with excess extracellular K+ and the application of caffeine induced a transient and dose-dependent elevation of the cytosolic free calcium concentration, with durations of 4 and 2 min, respectively. Transient elevations of calcium repeatedly appeared in response to both repetitive depolarization (100 mM K+) and caffeine (10 mM) applications with progressive reductions in peak levels. In either case, the fifth or later treatments induced little or no rise in levels of the cytosolic calcium. The amount of released calcium induced by high K+ depolarization after (n-1) time applications (1 less than or equal to n less than or equal to 5) of caffeine was equal to that induced by the n-th application of caffeine. The amount of released calcium induced by caffeine after (n-1) time exposures (1 less than or equal to n less than or equal to 5) to K+ depolarization was equal to that observed during the n-th exposure to K+ depolarization. These results indicate that caffeine- and depolarization-sensitive intracellular calcium storage sites may be identical and that caffeine and K+, in optimal concentrations, will release an equal amount of calcium from the same storage site in cultured arterial smooth muscle cells, irrespective of the amount of stored calcium.     

Steady-state kinetic properties of phosphoglycerate kinase of mung beans

Steady-state kinetics of the action of mung bean phosphoglycerate kinase have been investigated using 3-phosphoglycerate and ATP as substrates in the presence of Mg2+ ions. Keeping a constant and high Mg2+ concentration and varying the concentration of one of the substrates (ATP or 3-phosphoglycerates) at several fixed concentrations of the other substrate (3-phosphoglycerate or ATP), the Km values of Mg.ATP2- and 3-phosphoglycerate were found to be 0.42 and 0.60 mM, respectively. These values are independent of the concentration of the other substrate. A limiting value of Vmax, where the enzyme is saturated with both the substrates, was found to be 39.4 mumoles product formed per min per mg enzyme protein. This corresponds to a turnover number equal to 31.5 sec-1 (for molecular weight of the enzyme equal to 48,000). If [Mg2+] and [ATP4-] are held equal and varied together at several fixed concentrations of 3-phosphoglycerate, deviations from Michaelis-Menten kinetics (non-linear Lineweaver-Burk plots) are observed at lower values of ATP4- and Mg2+ (less than 0.1 mM), giving rise to apparent sigmoidicity in the rate versus [ATP4-] plots. It has been suggested that the real substrate for this enzyme is the Mg.ATP2- complex (and not free ATP4-). The complex dissociates at lower values of [Mg2+] and [ATP4-]. The resulting disproportionate decrease in the concentration of the complex brings about a steeper fall in the rate of reaction than is required by the Michaelis-Menten equation, giving rise to an apparent sigmoidicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of primary and secondary sites of parvalbumins using the two paramagnetic probes Gd (III) and Mn (II)

The binding of cations by parvalbumins was studied by the proton relaxation enhancement (PRE) method using the paramagnetic probes Gd(III) and Mn(II). Gd(III) appears as a specific probe of the primary sites CD and EF with the following binding parameters: n = 2, KdGd = 0.5 x 10(-11) M and epsilon b = 2.3. The low value of epsilon b is the result of a nearly complete dehydration of the protein bound ions. Competition experiments between Gd(III) and various diamagnetic cations show the following order of affinity for the EF and CD sites: Mg2+ less than Zn2+ less than Sr2+ less than Ca2+ less than Cd2+ less than La3+ less than or equal to Gd3+. Mn 2+ is a specific probe of a secondary site with the following binding parameters: n = 1, KdMn = 0.6 x 10(-3) M and epsilon b = 17. The high value of epsilon b suggests that the protein bound Mn(II) has retained most of its hydration shell. Competition experiments between (Mn(II) and different cations show similar affinities for this site: Ca2+ less than or equal to Mg2+ less than or equal to Cd2+ less than or equal to Mn2+. This secondary site is located near the EF primary site.     

The lysis effect of bull spermatozoa on gelatin substrate film methodical investigations.

Proteolytic activity in the acrosomes of ejaculated bull spermatozoa was demonstrated using an autoradiographic film as a gelatin substrate. Incubation of the spermgelatin adducts at +37 degrees C and 94% humidity, which was kept constant by ventilating an incubator with water-saturated compressed air, yielded reproducible results. Gelatin depolymerisation started adjacent to the posterior segment of the acrosome within 30 to 60 s after application of individual spermatozoa to the substrate membrane and, finally, increased to a white circular digestion area enveloping the entire sperm head. The observed gelatinolysis seems to be mainly caused by acrosin, the trypsin-like acrosomal proteinase. This conclusion is supported by the positive correlation (r = +0.83, P is less than or equal to 0.01) found between the mean values of the lysis areas of individual spermatozoa on gelatin films and the acrosin activity of the sperm population measured with Bz-Arg-OEt as substrate after acidic extraction of the spermatozoa. In addition, prior saturation of the substrate layers with acrosin inhibitor (SSPI-I, II) from boar seminal plasma prevented the lysis reaction. Extraction of acrosin from the spermatozoa before application to the gelatin membranes resulted in a complete loss of any proteolytic activity. If spermatozoa were stored for 4 to 6 days at +4 degrees C or -20 degrees C in Tris buffer and afterwards applied to the substrate layer, lysis areas of individual spermatozoa differed markedly. Spermatozoa from undiluted ejaculated frozen at -20 degrees C showed no proteolytic effect on gelatin films. In general, there was a high correlation (r = +0.83, P is less than or equal 0.01) between the number of "living cells" characterized by live-dead staining and the percentage of spermatozoa active on the substrate membranes.     

Quantum thermodynamics approach to phosphorylation and heterotrophic growth yields

A model of cell growth is presented which is based on the double postulates of quantized loss of energy during phosphorylation and reversible biosynthesis of cell structure. An immediate consequence of the postulates is the identical value for the energy efficiency of the phosphorylation and for that of the whole growth process. Another consequence is the relationship between the energy level of the biomass and the phosphorylation potential as embodied in the equation: EO = gamma'M X EATP, where EO is the heat of transfer of a pair of electrons to oxygen, EATP, the molar heat of hydrolysis of ATP, and gamma'M, the degree of reduction of the biomass, gamma M being constant and equal to 5. The model predicts five levels of growth yields corresponding to five permissible values for the P/O ratio (r = 0, 1, 2, 3, and 4). Any growth process would be characterized by a set of two integers N and lambda; N is the maximal P/O ratio prescribed by the energy content of the substrate as compared with that of the biomass, and lambda the number of further downward quantum jumps of the P/O ratio resulting from the adversity of the growth condition (N - lambda = r). Under full aerobiosis, one has 0 less than or equal to lambda less than or equal to N less than or equal to 3. When growth is limited only by the energy content of the substrate (lambda = 0), the time-independent dispersion of N, owing to substrate-level phosphorylations and (or) dephosphorylations, leads to effective values which are higher than the nominal ones for the yield per mole of oxygen and the heat of transfer of a pair of electrons. Under adverse conditions (lambda greater than 0), the apparent variations of the yields and the P/O ratio in function of the growth rate are shown to be an effect of the random dispersion of lambda and of the existence of a maximal rate of substrate consumption. Statistical evidence for the macroscopic quantum effect in heterotrophic growth is presented.     

Structure of calmodulin refined at 2.2 A resolution

The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.