Shape and thermodynamic parameters of a Ca2+-dependent ATPase. A solution x-ray scattering and sedimentation equilibrium study

Solution X-ray scattering and sedimentation equilibrium experiments in solvents of variable density were used to determine some thermodynamic parameters of deoxycholate-solubilized ATPase monomers. Molecular weight and partial specific volume of the unsolvated deoxycholate-ATPase complexes were determined from X-ray scattering and/or sedimentation equilibrium data in $\text{H}{}_2\text{O}\text{H}{}_2$¹⁸O mixtures. Volume, mean density (electron density) and hydration were determined by analysis of both solution X-ray scattering and sedimentation equilibrium data recorded in solvents containing various concentrations of sucrose. Protein shape was investigated further with the help of the autocorrelation function corresponding to the scattering curve recorded in the absence of sucrose. This curve was shown to represent mainly the particle shape. The autocorrelation function is fitted adequately by a model formed by two adjacent and coaxial cylinders of different heights and diameters. One cylinder is assumed to bind deoxycholate, while the other one, which is larger, is considered to represent the portion of the ATPase molecule that, in sarcoplasmic reticulum membranes, is exposed to the outer medium.     

Reversible redox titrations of cytochrome c and cytochrome c oxidase using detergent solubilized electrochemically generated mediator-titrants

The repetitive, $\text{reversible}$ equilibrium redox cycling of cytochrome $\text{c}$, cytochrome $\text{c}$ oxidase, or mixtures thereof has been made possible by the use of the oxidant, ferricinium ion. This ion is electrochemically generated by the use of non-ionic detergent solubilized ferrocene which is apparently incorporated as micelles and readily electron transfers with an electrode. The $\text{ferricinium-ferrocene}$ couple equilibrates rapidly with these heme proteins. Electrochemically generated benzylviologen radical cations are used as the reductant. The E^O′ values for cytochrome $\text{c}$ oxidase at pH 7.0 are 209 ± 15 mv (2e^?) and 340 ± 15 mv (2e^?).     

Detection and resolution of closely related satellite DNA sequences by molecular cloning.

Highly repeated satellite DNAs often consist of mixtures of DNAs with closely related repeating sequences. By cloning individual molecules we have resolved the 1.705 g/cm³ satellite DNA of Drosophila melanogaster into two distinct components: poly$\text{d}\text{A-A-G-A-G}\text{T-T-C-T-C}$ and poly$\text{d}\text{A-A-G-A-G-A-G}\text{T-T-C-T-C-T-C}$. The presence of two distinct sequences within this physically homogeneous satellite DNA had not previously been detected by standard physical, chemical, or sequencing techniques. Both cloning and direct sequence analysis suggest that the five-base-pair and seven-base-pair repeating units reside on separate molecules and are not interspersed with each other.     

Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.     

Studies on the molecular weight of the adenovirus type 2 hexon and its subunit

The molecular weight of the adenovirus type 2 hexon was calculated from sedimentation equilibrium, light scattering and sedimentation and diffusion experiments. The extinction coefficient, E_{1 cm}^1%, was determined to be 14.3 at 279 nm, from quantitative nitrogen and carbon analyses combined with the N,C content calculated from the amino acid composition. Other parameters determined were: the partial specific volume, $\text{}\text{?}\text{gn = 0.738}\text{cm}{}^3\text{g}{}^{\mathrm{?1}}$; the refractive index increment, $\text{(}\text{?n}\text{?c}\text{)}{}_{\mathrm{<mtext>T,P</mtext><mtext>,\mu </mtext>}}\text{= 0.193}\text{cm}{}^3\text{g}{}^{\mathrm{?1}}$ at 435.8 nm; the sedimentation coefficient, s_20,w⁰ = 13.0 S; and the diffusion constant, D_{20, w}⁰ = 3.32 × 10^?7 cm² s^?1. All molecular weights were between 355,000 and 363,000. Crystal density measurements were made on native and glutaraldehyde cross-linked crystals and the molecular weights calculated from these data were compared with the precise molecular weight determined by physico-chemical methods.Only one polypeptide of molecular weight 120,000 was found in reduced, or reduced and alkylated, hexon. Four or six organomercurial molecules were bound per 120,000 molecular weight of native hexon upon titration with 2-chloromercuri-4-nitrophenol and 2-chloromercuri-4,6-dinitrophenol, respectively. With 5,5′-dithiobis (2-nitrobenzoic acid) only one SH-group per 120,000 could be titrated in native hexon, but after denaturation in 1% sodium dodeeyl sulphate five more SH-groups reacted per 120,000 molecular weight. Thus there are three identical polypeptides of molecular weight 120,000 per hexon of total molecular weight 360,000.     

Some characteristics of monolayers of 1-palmitoyl-2-oleoyl-phosphatidylglycerol with and without dipalmitoylphosphatidylcholine during dynamic compression and expansion

Some properties of monolayers of $\text{1-}\text{palmitoyl}\text{-2-}\text{oleoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phospho}\text{-rac-}\text{glycerol}$ (POPG) alone or of POPG in mixtures with $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) have been measured near 35°C during dynamic compression and expansion at 3.6 cm²·s^?1. (2) The mean values of minimum surface tension (corresponding to maximum surface pressure) which could be obtained with pure POPG monolayers at high compression ranged from 15 to 18 mN·m^?1 in the presence of Na⁺, Ca²⁺ or low pH (2.0) in the subphase. (3) The presence of Ca²⁺ or low pH in the subphase increased the collapse plateau ratios obtained on cyclic compression. This might represent enhanced respreading into the monolayer of pure POPG from a collapsed form during reexpansion of the surface. (4) Monolayers containing 10% or 30% POPG and 90% or 70% DPPC could be compressed to surface tensions approaching zero. (5) In such mixed monolayers, 10% or 30% POPG did not appear to enhance respreading, as measured by collapse plateau ratios, in the presence of Na⁺ or Ca²⁺ in the subphase.     

Gelation of sickle cell hemoglobin in mixtures with normal adult and fetal hemoglobins.

We report the results of thermodynamic and kinetic studies on the gelation of mixtures of sickle cell (S) deoxyhemoglobin with normal human adult (A) and fetal (F) deoxyhemoglobins. The delay time of thermally induced gelation was monitored by the increase in turbidity. At the completion of gelation the solubility was determined by sedimenting the polymers and measuring the supernatant concentration spectrophotometrically. Addition of hemoglobins A or F, at mole fractions from 0 to 0.6, resulted in large increases in both the solubility and the delay time. For a 50:50 mixture of deoxyhemoglobin F with deoxyhemoglobin S, the solubility increased by a factor of 1.8 and the delay time by a factor of 10⁷ relative to pure deoxyhemoglobin S at the same total concentration, while for a 50:50 mixture of deoxyhemoglobins A and S the solubility increased by a factor of 1.4 and the delay time by a factor of 10⁴. The relative delay times were independent of both temperature and total hemoglobin concentration. The data have been analyzed according to theoretical models which treat the effects of temperature, concentration, non-ideality and solution composition on the thermodynamics and kinetics of gelation. The increased solubility in mixtures with deoxyhemoglobin F is fully explained by a model in which only deoxyhemoglobin S molecules polymerize. The effect of fetal hemoglobin (α₂γ₂) and hybrid α₂γβ^S molecules is to increase the solution non-ideality through the contribution of their excluded volume. The smaller increase in the solubility observed in comparable mixtures with deoxyhemoglobin A requires that the hybrid α₂β^Aβ^S molecules copolymerize with the deoxyhemoglobin S. The kinetic results for the mixtures can be quantitatively accounted for using a nucleation model in which the equilibrium properties of the polymer are used to describe the critical nucleus. The very large increases in delay time observed for the $\text{S}\text{F}$ mixtures can be explained by assuming that only α₂β₂^S molecules participate in the formation of a nucleus containing about 25 monomers. As in the thermodynamic analysis, the smaller effect of adding deoxyhemoglobin A can be attributed to the contribution of the hybrid molecules in forming the critical nucleus. Thus the difference between the polymerization properties of mixtures of deoxyhemoglobin S with deoxyhemoglobins A and F can be attributed solely to the copolymerization of the α₂β^Aβ^S hybrid molecule and the absence of any significant copolymerization of the α₂γβ^S hybrid.     

Genetic changes in yeast cells cotransformed with mitochondrial DNA and plasmid YEp13 are not elicited by recombinant molecules made by covalent insertion of mitochondrial DNA into YEp13

$\text{Saccharomyces cerevisiae}$ strain DC5-E2 that lacks mtDNA ($\text{leu2 rho}{}^{\mathrm{o}}$) can be cotransformed with a mixture of mtDNA and the plasmid YEp13 (LEU2/2μ/pBR322) to produce Leu⁺ transormants which, on being mated to $\text{mit}{}^{\mathrm{?}}$ tester strains, generate respiratory competent diploids (such events are denoted marker rescue). In this work strain DC5-E2 was transformed with recombinant molecules consisting of a mtDNA segment including the $\text{oli2}$ gene inserted into YEp13. The Leu⁺ transformants made with the recombinant plasmids were unable to rescue $\text{mit}{}^{\mathrm{?}}$ testers carrying mutations in the $\text{oli2}$ region, in contrast to Leu⁺ cotransformants made with mixtures of YEp13 and $\text{oli2}$ mtDNA. We conclude that marker rescue events occur as a result of interactions between the mtDNA of the $\text{mit}{}^{\mathrm{?}}$ tester and the mtDNA sequences introduced by transformation. Such interactions cannot occur when the latter mtDNA is forced to replicate in covalent association with YEp13, probably in the nucleus.     

Molecular weight distribution of interacting proteins calculated by multiple regression analysis from sedimentation equilibrium data: an interpretation of alpha s1-kappa-casein interaction.

Multiple regression analysis in four series with exponentially spaced molecular weight scales shifted by a factor of $\text{2}{}^{\mathrm{<mtext>1</mtext><mtext>4</mtext>}}$ between series was applied to sedimentation equilibrium data for determination of the molecular weight distribution of synthesized model systems consisting of up to three components which represented heterogeneous associating systems. Negative weight fractions of the components which were frequently encountered during multiple regression analysis were forced to zero by successively eliminating from the regression matrix the corresponding molecular weights in order of the magnitude of negative t values. The simplex optimization technique in conjunction with a prohibit-trespassing routine was used to maximize F values obtained from multiple regression analysis in search of the best fit values of component molecular weights. The quantitative information relating molecular weights and relative concentrations obtained by simplex optimization supplemented the regression matrix for calculation of the molecular weight distribution to improve the resolution of the molecular weight distribution patterns. This multiple regression method when carried out in conjunction with the simplex optimization provided a better fit to the data than the linear programming method of Scholte. When applied to mixtures of three standard proteins, the simplex optimization routine yielded values of molecular weights and relative concentrations of the component proteins which were in good agreement with the known values of the original mixtures. The molecular weight distribution calculated from sedimentation equilibrium data of α_s1-κ-casein mixtures using a uv scanner indicated that κ-casein readily dissociated to an oligomer, probably up to trimer, and interacted with the monomer or dimer of α_s1-casein forming a complex of approximately 400,000 molecular weight. This dissociation of κ-casein was promoted in the presence of ovalbumin, a nonmilk protein.     

Effect of pH,mono- and divalent cations on the mixing of phosphatidylglycerol with phosphatidylcholine. A monolayer (π, ΔV) and fluorescence study

The mixing of various molecular species of phosphatidylglycerol and phosphatidylcholine differing in their acyl chain lengths has been studied both in monolayers (π, Δ$\text{V}$), and in water dispersions (fluorescence polarization) with varying pH and ionic strength of the aqueous phase and in the presence of the divalent cations Mg²⁺ and Ca²⁺. In dilauroylphosphatidylglycerol/dipalmitoylphosphatidylcholine mixtures, both in monolayers and in water dispersions, no phase separation was detected at pH 2.9 where phosphatidylglycerol was protonated. With dipalmitoylphosphatidylglycerol/dipalmitoylphosphatidylcholine mixtures, in monolayers and at the same pH, no phase separation was detected for surface pressures below $\text{π = 40}\text{mN}\text{·}\text{m}{}^{\mathrm{?1}}$. In monolayers, and under ionic conditions such that phosphatidylglycerol was ionized (pH 5.6, 10 mM NaCl) miscibility was observed with dilauroylphosphatidylglycerol and dipalmitoylphosphatidylcholine and also with dipalmitoylphosphatidylglycerol and dilauroylphosphatidylcholine. Varying the ionic strength did not alter the miscibility of these lipids. The divalent cations Mg²⁺ and Ca²⁺ did not modify that of dilauroylphosphatidylglycerol with dilauroylphosphatidylcholine or with dipalmitoylphosphatidylcholine. Both in monolayers and in water dispersions, dipalmitoylphosphatidylglycerol and dilauroylphosphatidylcholine appeared to be at least partly miscible, in the presence of magnesium. Only in the presence of calcium and at high surface pressure might the monolayer data account for phase separation between these two lipids. The data presented demonstrate the existence of strong cohesive forces between phosphatidylcholine and phosphatidylglycerol with a marked influence of the former on the physical state of the latter. From an analysis of the Δ$\text{V}$ data, it is suggested that intrafacial hydrogen bonds may play a significant role in stabilizing phosphatidylcholine/phosphatidylglycerol mixtures.     

Structure and properties of hemocyanins. XIII. Dissociation of Helix pomatia alpha-hemocyanin at alkaline pH

Helix pomatia α-hemocyanin can dissociate stepwise into $\text{1}\text{2}$-size, $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules. Both $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules can occur in two isomeric forms, differing considerably in sedimentation behaviour.The effects of pH, ionic strength, temperature, Ca²⁺ concentration and oxygen pressure on these dissociation and isomerization steps were investigated systematically by sedimentation analysis.Dissociation and isomerization are favoured by increasing pH or temperature. Changes in ionic strength affect each step differently. Calcium ions are extremely effective in preventing dissociation and isomerization at low ionic strength, but this stabilizing ability diminishes at higher ionic strengths. Oxygen binding shifts the pH-dependent dissociation of whole into $\text{1}\text{2}$-size molecules to higher pH. Oxygen has no effect on the other dissociation steps. Intermolecular interactions appear to be predominantly electrostatic.     

Hydration of Escherichia coli lipids: Deuterium T1 relaxation time studies of phosphatidylglycerol,phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic $\text{1,2-}\text{dioleoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ in H₂O/²H₂O mixtures (9:1, v/v) were investigated with ²H-NMR. Comparison of the ²H₂O spin lattice relaxation time ($\text{T}{}_1$) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H₂O systems. Two distinct hydration regions could be discerned in the $\text{T}{}_1$ relaxation time profile. (1) A minimum of 11–16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of $\text{T}{}_1\text{≈ 90}\text{ms}$. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with $\text{T}{}_1\text{= 500}\text{ms}$ and hydration water with $\text{T}{}_1\text{= 80–120}\text{ms}$. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

Effect of protected protein supplements on some testicular traits in Brahman cross bulls

Differences in a number of testicular traits were examined in 12 Brahman cross (F₂ generation $\text{1}\text{2}$ and $\text{3}\text{4}$ BX) bulls fed either poor-quality native pasture (NP) hay or NP hay with a protected protein supplement. Supplementation for 60 days significantly (P < 0.05) increased roughage dry matter intake (7.7 $\text{v}$ 5.6 kg/head/day), enabling maintenance of liveweight, whereas control animals lost 40 kg. There were significant (P < 0.05) decreases in scrotal circumference (1.5cm) and testicular consistency (0.8 score) in the control group, in which testes weights at slaughter were significantly (P < 0.05) less (373 g $\text{v}$ 459 g), with corresponding lower epididymal weights (37.4 g $\text{v}$ 43.5 g). Estimates of daily sperm production per gram (DSPG) were similar for both groups, and testis daily sperm production (DSP) was somewhat but not significantly (P>0.05) lower in the control group (4.3 × 10⁹$\text{v}$ 6.0 × 10⁹) as a result of lower testis weights. Total epididymal sperm storage capacity was also lower in control bulls (17.2 × 10⁹$\text{v}$ 27.0 × 10⁹), but only significantly (P < 0.05) in relation to cauda sperm reserves (8.5 × 10⁹$\text{v}$ 13.6 × 10⁹). Luteinizing hormone (LH) and testosterone responses to gonadotrophin releasing hormone (GnRH) treatment were similar for both groups, although LH responses to GnRH were greater in $\text{1}\text{2}$ BX than in $\text{3}\text{4}$ BX bulls.     

Binding of steroids to P.testosteroniΔ5-3-ketosteroid isomerase: Measurement of the number of binding sites by equilibrium dialysis

The binding of progesterone, 17β-estradiol and 19-nortestosterone acetate to the Δ⁵-3-ketosteroid isomerase from $\text{Pseudomonas}$$\text{testosteroni}$ has been investigated by the technique of equilibrium dialysis. Under the conditions used, all three steroids formed 2:1 complexes with each molecule of enzyme dimer (M.W. = 26,788). No evidence of any cooperative binding phenomena was obtained. The dissociation constants of the enzyme steroid complexes at 25°C were: progesterone, 2.2 μ$\text{M}$; estradiol, 2.5 μ$\text{M}$; 19-nortestosterone acetate, 9.2 μ$\text{M}$.     

Interactions of proteins and cholesterol with lipids in bilayer membranes

Mixtures of lipids and proteins, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$, of the lipid and aggregated below $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$. For mixtures af dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains was shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present.In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50–200 nm in length, around smooth patches of lipid.Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperarture of the lipid is discussed.     

Aureobasidium pullulans metabolism of prostaglandins of the A-type

$\text{Aureobasidium pullulans}$, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA₂ (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9α or 9β-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9α, 15α-dihydroxy-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9β-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9,15-oxo-2,3,4,5-tetranorprostanoic acid and 9,15-oxo-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. The water soluble metabolites have not been characterized further.The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that $\text{A. pullulans}$ can perform the following transformations: β-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both Δ^10,11 and Δ^13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (Δ^10,11 → Δ^11,12). $\text{A. pullulans}$ metabolizes 15-epimeric PGA₂ equally readily with the production of similar products. PGA₁ affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. $\text{A. pullulans}$ displays some enantioselectivity, PGA₂ and 15-epi-PGA₂ are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in $\text{phosphocreatine}\text{creatine}$ ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in $\text{[}\text{phosphocreatine}\text{]}\text{[}\text{creatine}\text{]}$ ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N′-(4-dimethylamino-α-naphthyl)carbodiimide

The $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rabbit sarcoplasmic reticulum, when labelled at two Ca²⁺-protected sites with $\text{N-}\text{cyclohexyl}\text{-N′-(4-}\text{dimethylamino}\text{-α-}\text{naphthyl}\text{)}\text{carbodiimide}$ (NCD-4) retains Ca²⁺ binding capacity at the sites with $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values of approx. 3 μM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca²⁺ binding sites present in the native ATPase. The NCD-4 labelled ATPase exhibits slow conformational changes at each site on addition of Ca²⁺. It retains the ability to form phosphoenzyme, and can most likely translocate Ca²⁺.     

Transfection of Vibrio cholerae by bacteriophage phi 149 DNA

DNA isolated from Choleraphage ø149 of Group IV was infectious when mixed with competent $\text{V.}\text{cholerae}$ cells. The cells were competent during mid-log phase of growth. The infectivity of phage DNA was destroyed by deoxyribonuclease but not by ribonuclease or pronase. About 5 min is required for the establishment of the DNase resistant state. The dose response curve for transfection suggested that 2 to 3 molecules of DNA are required to produce one infections center. An infectivity of 5 × 10⁴ infectious center per μg of DNA was obtained.     

Alkaline phosphatase. A dominant enzyme of microvillus structure of cephalopod photoreceptors

The rhabdomeres of cephalopod photoreceptors, which are built up mainly of rhodopsin and phospholipid molecules, show a very high alkaline phosphatase activity. The enzyme has been partially characterized in purified rhodopsin vesicle fractions of the rhabdomeres by the following kinetic data: pH optimum 8.7; activation energy 9100 cal·m^?1; $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 2.5}$ μmol·min^?1·mg^?1; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 1.5·10}{}^{\mathrm{?4}}\text{M}$; its activity depends on Mg²⁺. There is good evidence that the alkaline phosphatase is a membrane-bound enzyme with receptor sites presumably located on the inside of the membrane. This enzyme has not been purified but its high activity compared to that of other known alkalin phosphatases (see Table I) indicates that each mirovillus, the structural unit of the rhabdomere, contains 1–20 enzyme molecules. This finding supports the hypothesis that the alkaline phosphatase is involved in the biochemical amplification process of excitation, or adaptation.