The effect of temperature and Ca2+ on the interaction of high-density lipoproteins with epithelial cells in the human small intestine

The effect of temperature and Ca2+ ions on the interaction of high-density lipoprotein (HDL3) with human enterocytes was studied. It was shown that Kd measured at 4 degrees C is similar to that at 37 degrees C. Maximal number of binding sites at 37 degrees C is 15-fold times higher than that at 4 degrees C. EDTA (10 mM) and CaCl2 (0.5-5 mM) did not affect binding and uptake of HDL3 by human enterocytes. The obtained results indicate that HDL3-binding with these cells depends on temperature and does not depend on Ca2+ ions.     

LcrG, a secreted protein involved in negative regulation of the low-calcium response in Yersinia pestis.

The purpose of this study was to define the function of LcrG, the product of the first gene in the lcrGVHyopBD operon of the low-Ca(2+)-response (LCR) virulence plasmid of Yersinia pestis. We created a Y. pestis strain having an in-frame deletion in lcrG. This nonpolar mutant had an abnormal LCR growth phenotype: it was unable to grow at 37 degrees C in the presence of 2.5 mM Ca2+ ("Ca2+ blind") but was able to grow at 37 degrees C when 18 mM ATP was present. At 37 degrees C it failed to downregulate the expression and secretion of its truncated product (LcrG), V antigen, and YopM. All of these mutant properties were complemented by plasmids carrying normal lcrG. However, a nonpolar lcrE mutation and an lcrH mutation (both also causing a Ca(2+)-blind phenotype) were not complemented in this way. The Y. pestis parent strain expressed LcrG at 37 degrees C in the presence and absence of Ca2+ and transported it to the medium when Ca2+ was absent. We identified two LCR-regulated loci, lcrD and yscDEF, required for this transport. Complementation analysis of the Y. pestis lcrR strain previously shown to lack the expression of LcrG showed that the loss of LcrG but not of LcrR caused the Ca(2+)-blind phenotype of that mutant. Taken together, the results show that LcrG is a negative regulator of the LCR, perhaps functioning in Ca2+ sensing along with LcrE.     

Calcium sparks in mouse ventricular myocytes at physiological temperature

In cardiac muscle, Ca2+ is released from the sarcoplasmic reticulum (SR) in units called Ca2+ sparks. Ca2+ spark characteristics have been studied almost entirely at room temperature. This study compares characteristics of spontaneous sparks detected with fluo 3 in resting mouse ventricular myocytes at 22 and 37 degrees C. The incidence and frequency of Ca2+ sparks decreased dramatically at 37 degrees C compared with 22 degrees C. Also, spark amplitudes and times to peak were significantly reduced at 37 degrees C. In contrast, spatial width and decay times were unchanged. During field stimulation, peak spatially averaged transients were similar at 22 and 37 degrees C, and experiments with fura 2 demonstrated that diastolic and systolic Ca2+ concentrations were unchanged. However, SR Ca2+ content decreased significantly at 37 degrees C. Restoration of SR Ca2+ by superfusion with 5 mM Ca2+ increased spark frequency but did not reverse the effects of temperature on spark parameters. Thus effects of temperature on spark frequency may reflect changes in SR stores, whereas changes in spark amplitude and rise time may reflect known effects of temperature on ryanodine receptor function.     

Unusual, virulence plasmid-dependent growth behavior of Yersinia enterocolitica in three-dimensional collagen gels

As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen Yersinia enterocolitica in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37 degrees C. Motility at 37 degrees C was abrogated in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVDelta yadA-carrying yersiniae formed loosely packed microcolonies at 37 degrees C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, we demonstrated Ca(2+)-dependent expression of this pYV-localized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions.     

Ca2+- and Mg-ATP-dependent shape change of human erythrocyte ghosts and triton shells

Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10(-7)-10(-6) M Ca2+-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10(-7)-10(-6) M Ca2+, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 degrees C were highly sensitive to Ca2+. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl2 (pH 7.4) at 0 degrees C, they gradually lost Ca2+ sensitivity within 60 min, but they recovered Ca2+ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 degrees C followed by washing with 1 mM MgCl2 (pH 7.4). The shape of these highly Ca2+-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 degrees C in the presence of 10(-7)-10(-6) M Ca2+. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 degrees C shrank immediately in the presence of 10(-6) M Ca2+ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384-390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca2+ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca2+.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.     

Consequences of Ca2+ deficiency on macromolecular synthesis and adenylate energy charge in Yersinia pestis.

A 37 but not 26 degrees C virulent Yersinia pestis is known to require at least 2.5 mM Ca2+ for growth; this requirement is potentiated by Mg2+. After shift of log-phase cells (doubling time of 2 h) from 26 to 37 degrees C in Ca2+-deficient medium, shutoff of net ribonucleic acid synthesis preceded that of protein and cell mass. With 2.5 mM Mg2+, about two doublings in cell mass and number occurred before restriction with synthesis of sufficient deoxyribonucleic acid to account for initiation and termination of two postshift rounds of chromosome replication. Temperature shift with 20 mMMg2+ resulted in a single doubling of cell mass and number with one round of chromosome replication. Subsequent to shutoff of ribonucleic acid accumulation, ribonucleoside but not deoxyribonucleoside triphosphate pools became reduced to about 50% of normal values and the adenylate energy change fell from about 0.8, typical of growing cells, to about 0.6. Excretion of significant concentrations of adenine nucleotides under both permissive and restrictive conditions was observed. Only trace levels (less than 0.01 microM ol/g [dry weight]) of guaninosine 5'-diphosphate 3'-diphosphate accumulated under restrictive or permissive conditions; guanosine 5'-triphosphate 3'-diphosphate was not detected. Return of fully restricted cells from 37 to 26 degrees C with Ca2+ resulted in prompt growth, whereas addition of Ca2+ at 37 degrees C was ineffective. This finding indicates that the observed temperature-sensitive lesion in ribonucleic acid synthesis that results in restriction can be prevented but not reversed by cultivation with Ca2+.     

Effects of a surfactant-associated protein and calcium ions on the structure and surface activity of lung surfactant lipids

Previous studies have demonstrated that lung-specific proteins are associated with surfactant lipids, particularly the highly surface active subfraction known as tubular myelin. We have isolated a surfactant-associated protein complex with molecular weight components of 36 000, 32 000, and 28 000 and reassembled it with protein-free lung surfactant lipids prepared as small unilamellar liposomes. The effects of divalent cations on the structure and surface activity of this protein-lipid mixture were investigated by following (1) the state of lipid dispersion by changes in turbidity and by electron microscopy and (2) the ability of the surfactant lipids to form a surface film from an aqueous subphase at 37 degrees C. The protein complex markedly increased the rate of Ca2+-induced surfactant-lipid aggregation. Electron microscopy demonstrated transformation of the small unilamellar liposomes (median diameter 440 A) into large aggregates. The threshold Ca2+ concentration required for rapid lipid aggregation was reduced from 13 to 0.5 mM by the protein complex. This protein-facilitated lipid aggregation did not occur if Mg2+ was the only divalent cation present. Similarly, 5 mM Ca2+ but not 5 mM Mg2+ improved the ability of the protein-lipid mixture to form a surface film at 37 degrees C. Extensive aggregation of the surfactant lipids without protein by 20 mM Ca2+ or 20 mM Mg2+ did not promote rapid surface film formation. These results add to the growing evidence that specific Ca2+-protein-lipid interactions are important in determining both the structure and function of extracellular lung surfactant fractions.     

Calcium-induced membrane metabolic alterations modify the sex steroids binding into dog brain synaptosomal plasma membranes

The binding of 45Ca2+ into synaptosomal plasma membranes (SPM) of dog brain follows a sigmoid path. In graphical analysis of this binding the mean Hill coefficient (h) was 1.64 +/- 0.09 (r2 = 0.96 +/- 0.02). Binding of Ca2+ into SPM was saturable, with an apparent binding constant of 1.2 +/- 0.1 microM. At saturation, such calcium specific binding sites corresponded to 11.2 +/- 0.9 nmol/mg SPM protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of calcium into SPM of at least one class of high affinity specific binding sites. [14C]estradiol, [14C]estrone and [14C]progesterone, when incubated with SPM up to a concentration of 10 microM for 2 hr at 37 degrees C, bind into SPM at nmolar concentrations. Ca2+ ions up to 5 mM considerably increase steroids binding into SPM. This effect of calcium was concentration-dependent, reached saturation at approx 4-5 mM. Once calcium has promoted steroids binding, the subsequent addition of 25 mM EGTA failed to displace bound steroids. Molecular interactions between calcium and SPM was assessed by measuring the steady-state fluorescence polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), and by estimating the production of malondialdehyde (MDA) during 2 hr incubation of Ca2+ (5 mM) with SPM at 37 degrees C. The effect of Ca2+ on the SPM structure was to increase both the rigidity of the membrane and the MDA production. Chelation of Ca2+ (5 mM) with EGTA (25 mM) did not reverse the increase in the rigidity owing to metabolic alterations of SPM lipids (e.g. production of MDA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Adsorption of sphingomyelinase of Bacillus cereus onto erythrocyte membranes

Sphingomyelinase of Bacillus cereus proved to be specifically adsorbed onto mammalian erythrocyte membranes in the presence of either Ca2+ or Ca2+ plus Mg2+ in the order of sphingomyelin content; i.e., sheep, bovine greater than porcine greater than rat erythrocytes. No appreciable adsorption was observed in the presence of Mg2+ alone nor in the absence of divalent metal ions. The enzyme adsorption onto bovine erythrocytes was dependent upon the incubation temperature. By shifting the temperature from 37 to 0 degrees C, sphingomyelinase once adsorbed onto the surface of bovine erythrocytes was released into the supernatant. Ca2+ proved to be an essential factor for the enzyme adsorption: The addition of 1 mM Ca2+ enhanced the adsorptive process, but inhibited sphingomyelin hydrolysis and hot or hot-cold hemolysis of erythrocytes, while the addition of 1 mM Ca2+ plus 1 mM Mg2+ enhanced sphingomyelin breakdown and hemolysis as well as the enzyme adsorption. However, when the amount of sphingomyelin fell off to 0.2-0.7 nmol/ml or less by the action of sphingomyelinase, the enzyme once adsorbed was completely released from the surface of erythrocytes. The result indicates that the major binding site for sphingomyelinase is sphingomyelin. In the presence of 1 mM Mg2+ alone, the enzymatic hydrolysis of sphingomyelin and hemolysis proceeded whereas the enzyme adsorption was not encountered during 60 min incubation at 37 degrees C. The change in the molar ratio of Ca2+ to Mg2+ affected the enzyme adsorption and sphingomyelin breakdown; the higher Ca2+ enhanced the adsorption whereas the higher Mg2+ stimulated sphingomyelin hydrolysis.     

A kinetic investigation of the effects of adrenaline on 45Ca2+ exchange in isolated hepatocytes at different Ca2+ concentrations, at 20 degrees C and in the presence of inhibitors of mitochondrial Ca2+ transport.

The effects of adrenaline on 45Ca2+-exchange curves for isolated hepatocytes incubated under various steady-state conditions were investigated. Kinetic analysis showed that the simplest compartment configuration consistent with each set of data was a series configuration of a three-compartment closed system comprising compartment 1 (C1), the extracellular medium, and two kinetically distinct compartments of cellular exchangeable Ca2+, C2 and C3 (C1 = C2 = C3). Subcellular fractionation of hepatocytes labelled with 45Ca2+ at 0.1 mM-Ca2+ indicated that C3 includes exchangeable Ca2+ in the mitochondria and endoplasmic reticulum. The following results were obtained from experiments conducted at 37 degrees C at five different extracellular Ca2+ concentrations. For both untreated and adrenaline-treated cells, plots of the flux from C1 to C2 as a function of the extracellular Ca2+ concentration were best described by straight lines consistent with Ca2+ influx across the plasma membrane being a diffusion process. Adrenaline increased the value of the permeability constant for Ca2+ influx by 40%. For untreated cells, plots of the flux between C2 and C3 as a function of the concentrations of Ca2+ in these compartments approached a plateau at high Ca2+ concentrations. Adrenaline caused a 3-fold increase in the concentration of Ca2+ that gives half-maximal rate of Ca2+ transport from C2 to C3. At 1.3 mM extracellular Ca2+, a decrease in incubation temperature from 37 degrees C to 20 degrees C decreased the quantity of Ca2+ in C3 and the flux and fractional transfer rates for the transport of Ca2+ between C2 and C3. At 20 degrees C adrenaline increased the quantity of Ca2+ in C3 and the fractional transfer rates for the transfer of Ca2+ from C1 to C2, and from C2 to C3. At 37 degrees C and 2.4 mM extracellular Ca2+, antimycin A plus oligomycin decreased the quantity of Ca2+ in C3 and increased the fractional transfer rate for the transport of Ca2+ from C3 to C2. In the presence of antimycin A and oligomycin, adrenaline did not increase the quantity of Ca2+ in C2 or the flux and fractional transfer rate for the transport of Ca2+ from C1 to C2, whereas these parameters were increased in the absence of the inhibitors.     

The endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells is Ca(+2)-independent and distinct from a Ca(+2)-dependent hyaluronan binding activity.

Isolated and cultured rat liver sinusoidal endothelial cells (LECs) retain the ability to specifically bind 125I-hyaluronan (HA) and internalize it using a coated pit pathway [Biochem J, 257:875-884, 1989]. Here we have determined the effect of Ca+2 on the binding and endocytosis of HA by LECs. 125I-HA binding to intact LECs at 4 degrees C occurred both in the absence (10 mM EGTA) or the presence of physiologic concentrations of Ca+2 (1.8 mM). However, the specific binding of 125I-HA to LECs increased linearly with increasing Ca+2 concentrations. After permeabilization with the nonionic detergent digitonin, the Ca(+2)-independent HA binding activity increased approximately 743%, while the Ca(+2)-dependent binding activity was enhanced only approximately 46%. Therefore, the Ca(+2)-dependent HA binding activity appears not to be intracellular, whereas the Ca(+2)-independent HA receptor is found both inside LECs and on the cell surface. When LECs were allowed to endocytose 125I-HA at 37 degrees C in 10 mM EGTA or in 1.8 mM Ca+2, no differences were seen in the extent or rate of endocytosis. When LECs were allowed to endocytose 125I-HA in the presence of 10 mM Ca+2, the amount of cell-associated radioactivity increased approximately 20-50-fold. However, this additional cell-associated 125I-HA was not sensitive to hyperosmolarity and was removed by washing the cells in 10 mM EGTA at 4 degrees C. Therefore, the Ca(+2)-dependent cell-associated 125I-HA had accumulated on the cell surface and had not been internalized. From these studies we conclude that LECs have at least two types of specific HA binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noninvolvement of the heat-induced increase in the concentration of intracellular free Ca2+ in killing by heat and induction of thermotolerance

Mouse C3H 10T1/2 cells exhibited a two- to threefold increase in the concentration of free Ca2+ during heating at 45 degrees C. The increase was maximal for a heat dose which was still in the shoulder region of the survival curve. The increase was fully reversible in heat-sterilized cells. By changing the concentration of extracellular Ca2+, it was possible to modulate the concentration of intracellular free Ca2+ in heated cells. Lowering the extracellular concentration to 0.03 mM reduced the baseline concentration of intracellular free Ca2+, and prevented it from increasing in heated cells to a level exceeding that of nonheated cells incubated in medium containing 2.0 or 5.0 mM Ca2+. Raising the concentration of extracellular Ca2+ to 15.0 mM raised the baseline, and resulted in a heat-induced increase in free Ca2+ which was twofold higher than that of cells heated in medium containing 2.0 or 5.0 mM Ca2+. An elevated concentration of intracellular free Ca2+ during and after heating did not potentiate thermal killing, nor did a reduced concentration during and after heating mitigate killing. Furthermore, the data argue against a heat-induced increase in free Ca2+ to some threshold level, which potentiates cell killing by some other parameter. In addition, cells heat-shocked in either 0.03 or 5.0 mM extracellular Ca2+, and then incubated in the same concentration for 12 h at 37 degrees C, developed quantitatively similar amounts of tolerance to a second heating. The data suggest that the concentration of intracellular free Ca2+ does not play a critical role in thermal killing or the induction and development of thermotolerance.     

A folding pattern that is stable to thermal cycling is achieved by long term storage of recombinant human beta-casein with four extra N-terminals amino-acid residues at -20 degrees C

Studies have followed the turbidity (OD400 nm) of beta-casein (CN) as temperature (T) increased from 4 to 37 degrees C. Native non-phosphorylated beta-CN showed a turbidity increase above 25 degrees C and precipitated at about 22 degrees C in 5mM Ca+2. These patterns were reproducible upon T-cycling while those of recombinant beta-CN proteins are not. Here, a wild-type recombinant that was thermally stable after being frozen in solution and stored at -20 degrees C for a prolonged period of time was denatured with guanidine HCl and refolded by dialysis against buffer. This protein was again not stable to T-cycling. A recombinant mutant with four extra N-terminal amino acids was very stable to T-cycling, both with and without 5mM Ca+2. However, it was still much different than the native protein. These results indicate that there are probably many energy minima for this protein and emphasize the possibility of "chaperon-like" conditions for proper folding of human beta-CN.     

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.     

Calcium ion as a probe of the monovalent cation center of sodium, potassium ATPase

Sodium and potassium ion-transport adenosine triphosphatase from dog kidney was incubated with 0.4-2 mM Ca2+ at 23 degrees C for more than 2 min in the absence of monovalent inorganic cations, cooled to 0 degrees C, and phosphorylated from 1 mM Pi with 2.4 mM MgCl2. The resultant phosphoenzyme resembled that obtained by incubating the enzyme with K+ in place of Ca2+ in six respects. It was concluded that Ca2+ can occupy the monovalent cation-binding center for K+. The rate constant for release of Ca2+ from the dephosphoenzyme at 0 degrees C was 0.17 s-1. The rate of release from the phosphoenzyme was at least 7-fold slower. Phosphorylation stabilized the binding of Ca2+ to the enzyme in contrast to its destabilization of the corresponding K X enzyme complex. K-sensitive phosphoenzyme did not respond to free Ca2+. Thus Ca2+ was not easily accepted by nor released from the phosphoenzyme and would not be an effective substrate for transport. A selective barrier against Ca2+ between the monovalent cation binding center and the extracellular solution is proposed. Release of calcium from the dephosphoenzyme yielded a conformation that was not phosphorylated from Pi. The enzyme changed the conformation of its center for phosphorylation before or at the same time that it changed the conformation of its center for ion transport.     

Influence of EDTA and metal ions on a metalloproteinase from Pseudomonas fluorescens biotype I

The inactivation of a metalloproteinase from Pseudomonas fluorescens Biotype I with EDTA was investigated at 22 degrees C and 37 degrees C. At 22 degrees C proteolytic activity decreases linearly with time and an inactive apoenzyme is obtained by dialysis. Proteolytic activity can be restored with several metal-ions, Ca2+, Zn2+, Mg2+, Sr2+ and co2+ give the best results. Activity and substrate specificity are influenced by the metal-ions. Reactivation depends on the concentration of the metal-ions, optimum concentration is 1 mM for Ca2+ and 50 microM for Zn2+. The isoelectric point of the apoenzyme is around 8.0, this is about 0.3 pH-units lower than the isoelectric point of the native proteinase. At 37 degrees C inactivation follows first order kinetics and is irreversible because of autolysis as shown by a gel filtration-experiment.     

Binding strength of calcium ion to bovine alpha-lactalbumin

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.     

Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).