Biomedical subjects

K L Brown

Publications and source records attributed to K L Brown.

At least 73 records · Page 4Linked to original sources

PubMed1992

Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL.

The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dagAp4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dagAp4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2). We have developed an improved protocol for the renaturation of sigma factors that have been isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE.

Amino Acid Sequence↗

PubMed1992

Two promoters for the whiB sporulation gene of Streptomyces coelicolor A3(2) and their activities in relation to development.

Two transcripts corresponding to the whiB sporulation gene of Streptomyces coelicolor A3(2) that differed in length at their 5' ends by 164 nucleotides were identified by S1 mapping. Their presumptive promoters differed from each other; the more downstream, P2, resembled typical prokaryotic promoters (i.e., those recognized by the major form of RNA polymerase) at five of six positions in its -10 and -35 regions; and the more upstream, P1, was comparably similar, instead, to the previously described hrdDp1 promoter (M. J. Buttner, K. F. Chater, and M. J. Bibb, J. Bacteriol. 172:3367-3378, 1990) around -40, -10, and +1. In surface cultures of the wild-type strain, the abundance of transcripts from the weak P1 promoter showed no obvious correlation with the developmental stage, whereas transcripts from P2 were barely detectable until aerial mycelium was present and then became relatively abundant, consistent with the developmental role of whiB. Both types of transcript were detected during and, to a lesser extent, after rapid growth in liquid culture. In addition, both promoters were utilized in vitro by RNA polymerase purified from a liquid culture of S. coelicolor. Transcription from P1 and P2 was observed during surface culture in strains carrying mutations blocking aerial mycelium formation (bldA and bldB) or the formation of spores in aerial mycelium (whiA, whiB, whiG, and whiH). Thus, whiB transcription is not severely dependent on any of these developmental genes, among which whiG is the determinant of a putative sigma factor specific for, and crucial to, sporulation.

Base Sequence↗

PubMed1992

Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni.

Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure.

Amino Acid Sequence↗

PubMed1992

[Regulation of dimethylbenzanthracene-induced P1-450 gene expression in human lung tumor cells in culture].

Dimethylbenzanthracene (DMBA) stimulates expression of P1-450 gene in human lung tumor cells (ChaGo). A concentration and time-dependent increase in the level of P1-450 specific mRNA sequences has been observed in ChaGo cells treated with sublethal concentrations of DMBA. The methylation pattern of "-CCGG-" sequence of most of the coding region and of the 3' end of P1-450 gene is not affected by such DMBA treatment; DMBA-treatment of the cell induces hypomethylation of only the 5' end "-CCGG-" sequences of the gene. These results demonstrate that DMBA-treatment of ChaGo cells induced increased expression of gene can be correlated to the increased degree of site specific hypomethylation of the internal "-C-" residues of the "-CCGG-" sequences located at the 5' end of the P1-450 gene. However, it is also possible to have other molecular mechanism of regulation.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1991

Stabilization of thermally labile alkylcobalamins by a haptocorrin from chicken serum.

Binding of neopentylcobalamin and benzylcobalamin to the apoprotein of a haptocorrin from chicken serum has been demonstrated spectrophotometrically. The spectra of the protein-bound cobalamins strongly resemble those of base-on alkylcobalamins and show that when unbound these sterically hindered alkylcobalamins are only approximately 75% (benzyl) and 40% (neopentyl) base-on, at neutral pH and at 5 degrees C. The haptocorrin was found to stabilize the spontaneous thermal decomposition of the neutral species of benzylcobalamin and neopentylcobalamin by 470-fold (3.6 kcal) and 166-fold (3.0 kcal), respectively, relative to the protein-free species. After correction of the activation parameters for the thermal decomposition of the protein-free, neutral alkylcobalamins for the relative proportions of base-on and base-off species, the haptocorrin was found to stabilize the base-on species of both alkylcobalamins by 275- to 1400-fold (approximately 3.3 to 4.3 kcal). From the temperature dependence of the decomposition reactions, the enthalpies of activation are found to be essentially identical for the protein-free and protein-bound species of either cobalamin. Thus, stabilization of the thermal decomposition of these sterically hindered alkylcobalamins by haptocorrin is entirely due to entropic factors.

Animals↗

PubMed1991

Molecular recognition in the binding of vitamin B12 by the cobalamin-specific Intrinsic Factor.

Equilibrium constants (given as log K/M-1) have been determined at pH 7.4 and 4 degrees C for binding by porcine Intrinsic Factor (B12-binding protein from the gut, specific for the 'cobalamin' series of Co corrinoids) of vitamin B12 or cyanocobalamin (10.5), cyanocobinamide, alpha-ribazole and alpha-ribazole-phosphate (main fragments produced by cleaving off the 'cobalamin' side-chain, all less than or equal to 3), and cyanocobinamide in the presence of greater than or equal to 10(-9) M ribazole (5.6 and independent of ribazole concentration), i.e. ribazole catalyses the binding of the cobinamide. It is proposed that the specificity of Intrinsic Factor for the cobalamins depends on the presence of the ribazole fragment in the cobalamin side-chain to promote an essential change in conformation before the corrinoid fragment can be bound.

Animals↗

PubMed1991

Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter.

Two sigma factors, sigma 35 and sigma 28, direct transcription from the Bt I and Bt II promoters of the cryIA(a) gene of Bacillus thuringiensis; this gene encodes a lepidopteran-specific crystal protoxin. These sigma factors were biochemically characterized in previous work (K. L. Brown and H. R. Whiteley, Proc. Natl. Acad. Sci. USA 85:4166-4170, 1988; K. L. Brown and H. R. Whiteley, J. Bacteriol. 172:6682-6688, 1990). In this paper, we describe the cloning of the genes encoding these two sigma factors, as well as their nucleotide and deduced amino acid sequences. The deduced amino acid sequences of the sigma 35 and sigma 28 genes show 88 and 85% identity, respectively, to the sporulation-specific sigma E and sigma K polypeptides of Bacillus subtilis. Transformation of the sigma 35 and sigma 28 genes into B. subtilis shows that the respective B. thuringiensis sigma factor genes can complement spoIIG55 (sigma E) and spoIIIC94 (sigma K) defects. Further, B. thuringiensis core polymerase reconstituted with either the sigma 35 or sigma 28 polypeptide directs transcription from B. subtilis promoters recognized by B. subtilis RNA polymerase containing sigma E and sigma K, respectively. Thus, sigma 35 and sigma 28 of B. thuringiensis appear to be functionally equivalent to sigma E and sigma K of B. subtilis. However, unlike the situation for sigma K in B. subtilis, the homologous sigma 28 gene in B. thuringiensis does not result from a late-sporulation-phase chromosomal rearrangement of two separate, partial genes.

Amino Acid Sequence↗

PubMed1991

Limb reconstruction with vascularized fibular grafts after bone tumor resection.

Limb-salvage operations are being used with increasing frequency for patients with malignant bone tumors. For children, when a biologic reconstruction is desired, the choice is often between conventional and vascularized fibular grafts. An experimental study was performed in dogs to compare the two types of fibular grafts for bridging segmental defects in the radius and ulna. Twenty-six adult dogs were divided into two groups and studied at intervals of two, three, four, six, and 12 months after transplantation. The conventional grafts healed by creeping substitution i.e., they were first partially resorbed before new bone was laid down. In contrast, the vascularized fibulae maintained their normal structure and hypertrophied by subperiosteal new bone formation. The conventional fibulae eventually hypertrophied but much later than the vascularized grafts. The vascularized grafts were stronger at four and six months. Between six and 12 months, both grafts remodeled to resemble the size and shape of the forearm bones they were replacing. These experimental results have influenced the treatment of patients. Vascularized fibular grafts are ideal for diaphyseal defects greater than 10 cm long, especially in very young children, a poorly vascularized bed, or when bone healing is delayed by chemotherapeutic agents. To maximize hypertrophy, an external fixator is used to immobilize the graft rather than a plate, which acts as a stress shield.

Adult↗

PubMed1990

Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter.

A crystal protein gene of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel is transcribed in vivo from two overlapping promoters that are activated at different times during sporulation. We reported earlier (K. L. Brown and H. R. Whiteley, Proc. Natl. Acad. Sci. USA 85:4166-4170, 1988) that an RNA polymerase containing a sigma subunit with an apparent Mr of 35,000 can transcribe in vitro from the promoter utilized from early to midsporulation. We now report the isolation of an RNA polymerase containing a sigma subunit with an Mr of ca. 28,000; this polymerase activates transcription in vitro from the promoter used from mid- to late sporulation. This form of RNA polymerase also directs transcription in vitro from promoters preceding two other crystal protein genes and a gene coding for a spore coat protein. On the basis of a comparison of the four promoters, we propose the following consensus sequence for the -10 region recognized by RNA polymerase containing the Mr-28,000 sigma subunit: 5'-TNATANNaTGag-3'. No consensus sequence could be derived for the -35 region. When the N-terminal amino acid sequence of the sigma 28 polypeptide was aligned with the amino acid sequences of known sigma subunits, significant homology was found with the N terminus of the mature form of the sigma K subunit of RNA polymerase isolated from sporulating cells of Bacillus subtilis.

Amino Acid Sequence↗

PubMed1989

The effect of local controlled release of sodium fluoride on the stimulation of bone growth.

This study was performed to test the feasibility of using a biodegradable delivery system for the local controlled release of sodium fluoride as a mechanism for the stimulation of local bone growth. Sodium fluoride grains were mixed with poly-DL-lactic acid (PLA), and the mixture was then dissolved in acetone. After driving off the solvent, sheets of this material were rolled into rods 3.2 mm in diameter and 28.6 mm long. These were inserted into the intramedullary canals of the right femora of adult New Zealand White rabbits. A sham implant, made the same way but containing no fluoride, was inserted into the contralateral left leg. The effects of three different concentrations of sodium fluoride, 0.125 g NaF/g PLA, 0.250 g NaF/g PLA, and 0.500 g NaF/g PLA, were determined after a 4-week implantation period. In a second set of experiments, the concentration was fixed at 0.500 g NaF/g PLA with implantation durations of 4, 8, and 16 weeks. Expressed in terms of property ratios (experimental/control), bending strengths of the femora were significantly raised in most groups with the notable exception of Group 3 (0.500 g NaF/g PLA, 4 weeks). Cross-sectional area ratios in the distal femora were increased in all groups compared to right/left values for a normal unoperated group (p less than 0.05), with the highest ratio being 1.50 (SD = 0.44) for Group 4 (0.500 g NaF/g PLA, 8 weeks). Only Group 4 animals showed significant (p less than 0.05) increases in midshaft area ratio and none had responses proximally. Growth rates, measured by sequential fluorochrome labeling of bone followed the area ratio data and were only significant for the distal femora.

Animals↗

PubMed1989

Glycine binding to rat cortex and spinal cord: binding characteristics and pharmacology reveal distinct populations of sites.

Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 microM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 microM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by greater than 15-fold. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and beta-alanine. In contrast, D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 microM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 microM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

PubMed1988

Kinetics and activation parameters of the reaction of cyanide with free aquocobalamin and aquocobalamin bound to a haptocorrin from chicken serum.

The kinetics of the reaction of cyanide with free aquocobalamin (H2OCbl) and with aquocobalamin bound to a vitamin B12-binding protein (haptocorrin) from chicken serum (HC-H2OCbl) have been investigated as a function of temperature and pH. The mechanism of replacement of H2O from protein-bound and free H2OCbl is apparently the same and involves attack of both CN- (k1) and HCN (k2). The reactions with HC-H2OCbl are somewhat slower than those with free H2OCbl, k1 being 22-fold smaller and k2 7-fold smaller at 25 degrees C. The relatively small effect of the protein on the rate constants supports the view that the metal in HC-H2OCbl is readily accessible to solvent. Activation parameters suggest that the transition states for ligand substitution are stabilized by nucleophilic participation (at least by CN-) and that ligand substitution on the protein-bound cobalamin proceeds through more ordered, concerted transition states. The latter effect suggests that the Co-O bond of H2OCbl is strengthened upon binding to the haptocorrin.

Animals↗

PubMed1988

Heteronuclear NMR studies of cobalamins. 31P NMR observations of cobalamins bound to a haptocorrin from chicken serum.

A vitamin B12-binding protein (haptocorrin) from chicken serum has been purified to homogeneity by photodissociative affinity chromatography and characterized by gel electrophoresis and UV-visible spectrophotometry of its aquocobalamin, hydroxocobalamin, and cyanocobalamin complexes. The haptocorrin is a glycoprotein with a molecular mass of about 70 kDa and a protein moiety of about 40 kDa. 31P NMR resonances of the haptocorrin-cobalamin complexes are relatively broad singlets (with or without proton decoupling) shifted downfield by 0.7-1.0 ppm from the position of the free cobalamin resonances. From the line width data, the relaxation of the phosphorus nucleus is found to be dominated by chemical shift anisotropy with a very minor (13%) component from dipolar interaction with the two nearest neighbor protons. The rotational correlation time of the haptocorrin at 25 degrees C is estimated to be 85 ns and the activation energy for rotational correlation 3.9 +/- 0.3 kcal mol-1. The downfield shift of the 31P resonances of cobalamins upon binding to the haptocorrin cannot be due to hydrogen bonding phosphodiester moiety or displacement of the axial base by a group on the protein. Calculations also show that the downfield shift is very unlikely to be due to dipolar deshielding of the phosphorus nucleus by the ring current of an aromatic residue of the protein. It is concluded that the downfield shift of the 31P resonance must be due to sterically induced changes in phosphodiester conformation which may, or may not, involve steric compression of the axial Co-N bond.

Algorithms↗

PubMed1988

Angular homeostasis II: Pursuit of a moving target in a plane and some implications for cardiac teratology.

A model of cardiac ontogenesis is analyzed. It is cast in terms of the geometry of the pursuit of a linearly moving target by the growth of a chain of cells in the same plane, the pursuer, which at each step adjusts its direction of growth towards the current position of the target. The endpoint is the fusion between them, which can occur in 2 modes: either by the leading cell of the pursuer catching up with the target (pursuer-mediated fusion, or PMF) or by the target running into the preformed side of the pursuer (target-mediated fusion, or TMF). The causal specifications are the step size, the speed of the pursuer, the speed of the target, the restoration constant, and the initial direction of the pursuer; the outcome variables are the number of steps to fusion and the mode of fusion. The pattern of behavior is complicated, being more-or-less regular over large tracts of values, interspersed with abrupt, threshold-like changes that may generate a dichotomous pattern of inheritance despite a continuous gradation of genetic or other causes. The temporary abolition of the correction process (a change introduced to simulate the pattern of the effect of a teratogen) may delay fusion and suggest how a septum may fail to fuse, the ductus arteriosus to close, or an endocardial cushion to form. But the model also predicts that under certain plausible conditions, the "teratogen" would speed up fusion and hence perhaps offset a genetic predisposition to a congenital defect.

Heart Defects, Congenital↗

PubMed1988

Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes.

We report the isolation of an RNA polymerase from sporulating cells of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel that directs transcription from the promoter region of an insecticidal crystal protein gene. The core components of this RNA polymerase are associated with a polypeptide that has an apparent mass of 35 kDa. Neither RNA polymerase holoenzyme isolated from vegetative B. thuringiensis, nor the core derived from this enzyme, is capable of transcribing from the crystal protein gene promoter region; the addition of gel-purified 35-kDa polypeptide to the core reconstitutes the specific transcribing capability. The reconstituted enzyme does not direct transcription from the promoters for the ctc or spoVG genes of Bacillus subtilis; however, this form of RNA polymerase does direct transcription from a promoter for the 27-kDa crystal protein of B. thuringiensis subsp. israelensis and from a promoter for a 29-kDa polypeptide present in cuboidal crystals of B. thuringiensis subsp. kurstaki HD-1. We propose a tentative consensus sequence based on the alignment of the three B. thuringiensis promoters. This consensus sequence is different from consensus sequences reported for promoters recognized by enzymes containing other sigma subunits, suggesting that the 35-kDa polypeptide is an unusual sigma subunit.

Bacillus thuringiensis↗

PubMed1988

Use of a computer data file for storage of heat resistance data on bacterial spores.

The use of a computer data file to compile published heat resistance data on bacterial spores is described. Information relating to sporulation, heating and recovery of the spores, in addition to decimal reduction times at different temperatures has been stored. The data file was constructed using Minitab and an indexer program on a VAX 11/750 computer operating on VMS. Storage of the data in this way enables rapid manipulation and analysis of large quantities of information relating to heat resistance of bacterial spores.

Clostridium↗

PubMed1988

Mechanical and bone ingrowth properties of a polymer-coated, porous, synthetic, coralline hydroxyapatite bone-graft material.

CHAG, that is, porous hydroxyapatite hydrothermally converted from the calcium carbonate exoskeleton of a coral (genus Goniopora), has been shown to be effective as a scaffold for bone ingrowth. The large pores in the material, however, resulted in low compressive strengths. Compressive testing was performed to assess the changes in mechanical properties by coating the internal surfaces of CHAG with DL-PLA. Plugs of CHAG with thick (3:1 chloroform to DL-PLA by weight), medium (10:1), and thin (30:1) coatings as well as uncoated CHAG were then implanted transcortically in the proximal third of the diaphysis of rabbit tibiae to assess the in vivo response. The mechanical tests demonstrated significantly improved compressive strength, stiffness, and energy absorption for coated specimens compared with uncoated specimens. Coated specimens were not significantly different from canine tibial cancellous bone in strength and stiffness although they achieved only 36% of the energy absorption capacity. Specimens from rabbit tibiae were harvested at 3, 12, and 24 weeks for interface shear strength determination and contralaterally for histological and histomorphometric assessment. At 12 weeks, uncoated CHAG plugs developed an average ultimate interface shear stress of 26.7 MPa compared with 17 MPa for specimens with 30:1 coatings and 8 MPa for specimens with 10:1 and 3:1 coatings. At 24 weeks, there were no significant differences in shear stress between any of the specimens. Histomorphometric assessments showed that the ratio of area fraction of new bone to area fraction of new bone and void space increased from 68-70% for specimens with 3:1 and 10:1 coatings at 3 weeks to 85.5-89.5% at 24 weeks. In comparison, uncoated and 30:1 specimens had area fraction ratios of about 82% at 3 weeks and 93% at 24 weeks. Histologic sections demonstrated direct apposition of new bone to both the coating and the hydroxyapatite as well as degradation of the coating.

Animals↗

PubMed1987

4',6-Diamidino-2-phenylindole, a novel conformational probe of the sarcoplasmic reticulum Ca2+ pump, and its effect on Ca2+ release.

Sarcoplasmic reticulum vesicles were noncovalently labeled at micromolar concentrations with the polycationic fluorescent reagent 4',6-diamidino-2-phenylindole (DAPI), and changes in the fluorescence intensity of the membrane-bound dye associated with functions of the Ca2+ pump and Ca2+ release were investigated. It was found that 1) DAPI fluorescence changed in the [Ca2+] range in which high affinity Ca2+ binding to the Ca2+-ATPase takes place. The time course of the Ca2+-induced changes of DAPI fluorescence was essentially the mirror image of that of tryptophan fluorescence. 2) The fluorescence intensity of bound DAPI decreased upon increase of the intravesicular [Ca2+] by either ATP-dependent Ca2+ accumulation or incubation with millimolar Ca2+ in the presence of a calcium ionophore. 3) Upon induction of Ca2+ release by adding caffeine after the completion of Ca2+ uptake, DAPI fluorescence showed transient changes. Two classes of binding sites of the sarcoplasmic reticulum membrane for DAPI were clearly distinguishable: a high affinity site (Ka = 3.0 X 10(5) M-1) with a capacity of about 1 mol/mol of Ca2+-ATPase (8.0 nmol/mg of protein) and low affinity sites with about 20-fold lower affinity and 10-fold larger capacity. The partially purified Ca2+-ATPase showed similar characteristics of high affinity DAPI binding, suggesting that DAPI bound to its high affinity site on the Ca2+-ATPase monitors the enzyme conformational changes coupled with the events described above. The high affinity binding of DAPI to the enzyme led to an increase of the initial rate of Ca2+ uptake and the inhibition of Ca2+ release induced by caffeine or ionic replacement. These results suggest that the Ca2+-ATPase is involved in some steps of the Ca2+ release mechanism.

Animals↗