Delta endotoxin of Bacillus thuringiensis subsp. israelensis.

From Bacillus thuringiensis subsp. israelensis, a proteinase-resistant protein was purified which exhibited toxicity to larval mosquitoes and cultured mosquito cells, lysed erythrocytes, and was lethal to mice. To extract the protein, a sporulating culture of B. thuringiensis subsp. israelensis was treated with alkali, neutralized, and incubated with trypsin and proteinase K. It was then purified by gel filtration and DEAE column chromatography. Up to 240 micrograms of toxic protein was purified from 1 g (wet weight) of culture pellet. Two closely related forms of toxic protein were obtained: the 25a and 25b proteins. The two forms comigrated near 25,000 daltons in a sodium dodecyl sulfate-polyacrylamide gel, were serologically related, and showed similar partial protease digestion profiles, but were distinguishable by DEAE chromatography and nondenaturing polyacrylamide gel electrophoresis. Protein sequencing data indicated the 25b protein lacked the two amino acids at the amino terminus of the 25a protein. A Western blot enzyme-linked immunosorbent assay of alkali-solubilized proteins that were not treated with proteases suggested the toxic 25a and 25b proteins were proteolytically derived from a larger molecule of about 28,000 daltons. Alkali-solubilized proteins from an acrystalliferous strain of B. thuringiensis subsp. israelensis and from B. thuringiensis subsp. kurstaki failed to cross-react with antibodies to the 25a protein.     

Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin.

The conformational transitions of calcium binding protein parvalbumin III from carp muscle were studied by scanning calorimetry, potentiometric titration and isothermal calorimetric titration. Changes of Gibbs energy, enthalpy and partial heat capacity were determined. The removal of calcium ions by EDTA is accompanied by 1) a heat absorption of 75 +/- 10 kJ per mole of the protein, 2) a decrease in the Gibbs energy of protein structure stabilisation of about 42 kJ mol-1 and 3) a decrease in thermostability by more than 50 K. The protonation of the acidic groups leads to a loss of calcium followed by denaturation, while the pH of the transition strongly depends on calcium activity. The enthalpy and heat capacity changes at denaturation are comparable with the values observed for other compact globular proteins.     

Cytomegalovirus proteins. I. Polypeptides of virions and dense bodies.

Cytomegalovirus virions and dense bodies were purified by sucrose velocity and equilibrium centrifugation from the medium of fibroblasts infected with the strain AD169. The final virus preparations were purified more than 228-fold with respect to cellular proteins as determined by double-isotopic labeling and at least 1,600-fold on the basis of changes in the ratio of total protein to virus particles. The protein content of purified particles approximated that found for purified preparations of other herpesviruses. Twenty polypeptides ranging from 22,000 to greater than 230,000 molecular weight were detected in purified virus preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polypeptides of virions and dense bodies were allocated on the basis of analyses of preparations containing differing percentages of virions and dense bodies. Six polypeptides were represented predominantly or exclusively in virions, and four polypeptides were represented predominantly or exclusively in dense bodies, whereas the remainder appeared to be shared by both types of particles. Four polypeptides were glycosylated, and at least three of these appeared to be shared by both particles. Four polypeptides were glycosylated, and at least three of these appeared to be shared by both particle types. The protein composition of cytomegalovirus differs profoundly from that of herpes simplex virus.     

The protein phosphatases involved in cellular regulation. 2. Glycogen metabolism

The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261] were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCl), 2A1 (0.2 M NaCl) and 2A2 (0.28 M NaCl) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the alpha-subunit of phosphorylase kinase, were unaffected by inhibitor-1 and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 microM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2A1. These activities were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg X ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2...     

Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis.

Two proteins from parasporal crystals of Bacillus thuringiensis subsp. israelensis were purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. The larger of the two proteins (molecular weight, 68,000) was not cytolytic, whereas the smaller protein (molecular weight, 28,000) was highly cytolytic when assayed against rat erythrocytes. When these proteins were assayed against larvae of the yellow fever mosquito, Aedes aegypti, the larger protein was at least 100-fold more toxic than the smaller protein. Although proteolytic activity was not detected in solubilized crystals nor in purified protein preparations, the toxin (molecular weight, 68,000) was readily degraded to smaller, nontoxic molecules, even when maintained at 4 degrees C. Mixtures of the two purified proteins were significantly more toxic to mosquito larvae than was either protein alone. Thus, it is likely that both the mosquitocidal and the cytolytic protein play roles in the overall insecticidal action of the parasporal crystal produced by this bacterium.     

Outer membrane proteins of Escherichia coli. V. Evidence that protein 1 and bacteriophage-directed protein 2 are different polypeptides.

Protein 1 from the outer membrane of Escherichia coli K-12 and protein 2 from a phage PA-2 lysogen of the same strain were isolated by differential sodium dodecyl sulfate extraction and purified by ion-exchange and gel filtration chromatography. Rabbit antisera were prepared against these proteins and showed no cross-reaction between proteins 1 and 2. The proteins have the same N-terminal amino acid but show small yet significant differences in amino acid composition. The proteins were cleaved with cyanogenbromide in solvents containing both formic acid and trifluoroacetic acid. By comparing the cleavage in these solvents, it was established that protein 1 yielded 5 cyanogen bromide peptides, and the sum of the molecular weights of these was equivalent to the molecular weight of the uncleaved protein. Protein 2 yielded 4 cyanogen bromide peptides, none of which was identical to those of protein 1, and the sum of these peptides was also equivalent to the apparent molecular weight of the uncleaved protein. Significant differences were also observed when tryptic peptides from the two proteins were compared. These results indicate that protein 1 and the phage-directed protein 2 are distinct, different, and apparently homogeneous proteins.     

The secondary structure of gap junctions. Influence of isolation methods and proteolysis.

Paired intercellular transmembrane channels, termed connexons, comprised of hexameric assemblies of gap junction protein, were isolated and purified from rat liver by exploiting their resistance to either Sarkosyl detergent solubilization or alkali extraction. The secondary structures of the gap junction proteins prepared by these methods were compared by circular dichroism (CD) spectroscopy. Both the spectra and the calculated net secondary structures of the proteins obtained by the two isolation methods were different. The protein isolated by the Sarkosyl treatment was found to be approximately 50% alpha-helical, while protein isolated by alkali extraction had a lower helix content (approximately 40%). In both types of preparations, however, the helical content of the gap junction protein was sufficiently large to be consistent with an all-helical model for the membrane-spanning parts of the structure. CD spectroscopy was also used to examine the effects of proteolytic digestion of the cytoplasmic domain on the net secondary structure of the detergent-treated gap junction protein. The membrane-bound fragments had a slightly higher proportion of their residues that were alpha-helical in nature, suggesting that the transmembrane and/or intra-gap domains are indeed enriched in this type of secondary structure. This information constrains the range of models which can be realistically proposed for the channel structure.     

Characterization of protein kinases from Blepharisma intermedium.

Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.     

The reserve proteins in the cells of mature cotyledons ofLupinus albus var.Lucky. I. Quantitative ultrastructural study of the protein bodies

Summary A quantitative ultrastructural study of the protein bodies of the lupin cotyledonary cells was performed to determine the protein content per cell. Two kinds of protein body were observed by transmission and scanning electron microscopy whatever the cellular type within the cotyledon. Some of these were conventional spherical structures, entirely filled with a dense protein matrix, others exhibited one large or several small light inclusions within the dense matrix. Even when a few light areas contained globoids, the majority remained of unknown nature, but could not be considered proteinaceous since they never reacted with specific protein dyes. The reserve protein content per cell was determined by image analysis on two seeds (L₁ and L₂) selected because they had a markedly different total protein content. The volume occupied by the dense matrix of the intracellular protein bodies was considered representative of the reserve protein quantity. The protein content per cell increased from the periphery to the centre of the cotyledon in the two seeds studied. The protein content per cell of the L₂ seed was generally found to be greater than the L₁ seed, in particular in the abaxial zone where it was markedly higher. The difference in total protein content of the two seeds was demonstrated to be primarily due to a differential alveolation of the protein bodies. These results will be used to study the relationship between the protein content of the cotyledonary cells and their nuclear DNA content.     

The anion-transport protein of the human erythrocyte membrane. Studies on fragments produced by pepsin digestion.

We have studied the fragmentation by pepsin in 1 M-acetic acid of the erythrocyte anion-transport protein in erythrocyte membranes. The location of the fragments obtained was determined by radioiodinating the protein with the use of lactoperoxidase, and identifying the labelled peptides obtained in peptide "maps" of thermolysin digests of the fragments. Three of the fragments were found to be related overlapping products, and shared a common C-terminus. The major site of pepsin cleavage leading to the C-termini of these fragments was shown to be close to the major site of extracellular cleavage of the protein by proteinases active at a neutral pH. Another two fragments were isolated and shown to be derived from the C-terminal portion of the protein. No well-defined large radioactive fragments of the protein were solubilized from the membrane by pepsin in 1 M-acetic acid, the bulk of the radioactivity attributable to the anion transport protein being recovered in very small fragments that could not be resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Our results suggest that the polypeptide chain of the anion-transport protein emerges at the extracellular face of the membrane 8000-13000 daltons on the N-terminal side of the major site of extracellular cleavage of the protein by proteinases that are active at a neutral pH.     

Aging of human fibroblasts in vitro. Correlations between DNA synthetic ability and cell size.

In tobacco cell suspensions, protein synthesis and mitotic activity were inhibited by amino acid analogues: p-fluorophenylalanine (pFPA) or 5-methyltryptophan (5MT). After inhibition by pFPA, when the mitotic activity recovered in the presence of phenylalanine and casein hydrolysate, the time table of the mitotic phases was permanently altered. The inhibiting effects of 5MT were effectively reversed by tryptophan addition to the medium. Therefore 5MT was selected for reversible protein synthesis inhibition in partially synchronized cell suspensions. When cytokinin was added in a culture where protein synthesis was inhibited by 5MT, no mitosis was observed after the cells were transferred to a hormone-free medium and protein synthesis restored by tryptophan. Cytokinin must again be added in order to restore mitosis. Thus, the hormone effectiveness of cytokinins required that protein synthesis remained undisturbed. The effect of the protein synthesis inhibition by 5MT upon the metabolism of N⁶-benzyladenine was investigated: the intracellular concentration of this cytokinin was not altered, whereas the metabolic pool of its derivatives was quantitatively reduced.     

Cardiac alpha-crystallin. I. Isolation and identification

A water soluble protein, a major component of the cytosolic fraction of rat heart cells, was purified using either reverse phase HPLC or antibodies affinity chromatography procedures and characterized. The protein has an apparent Mr of 24 k, as judged by SDS-gel electrophoresis. Under non-denaturing conditions, however, the protein occurs as a homomultimer (Mr between 400 and 650 k) of the monomeric 24 kDa species and could be selectively enriched by fractionation of the cytosolic fraction on 10 to 40% sucrose gradients. Polyclonal antibodies, raised against the denatured 24 kDa protein, were used to investigate its tissue distribution. Besides the heart, where it is very abundant, the 24 kDa protein is expressed also in other red muscles and in kidneys, but was not detectable in stomach, thymus, liver, and brain. The amino acid composition of the protein and the partial amino acid sequence of various proteolytic fragments was determined. A search for homologies of the primary structure of known proteins has shown that the 24 kDa protein is strikingly similar, if not identical to alpha-B-crystallin. In fact, the two proteins were found to be indistinguishable also by immunological criteria. This study demonstrates that the lens protein alpha B-crystallin is a major cytosolic component of heart cells.     

Activation of C55-isoprenoid alcohol phosphokinase from Staphylococcus aureus. II. Biophysical studies.

Physicochemical techniques were used to investigate the activation of C55-isoprenoid alcohol phosphokinase by synthetic lecithins. Complexes of the enzyme with phospholipids were prepared using a method employing sodium dodecyl sulfate as a protein-solubilizing agent. Circular dichorism and the intrinsic fluorescence of the kinase were used as optical probes of protein conformation with these complexes. No evidence for a major lipid-dependent conformational change in the protein was observed when these complexes were studied under conditions where the lipid mesomorphic transitions occurred. EPR studies of mixtures of synthetic lecithins and the C55-isoprenoid alcohol indicated a correlation between kinase activity and the rotational diffusion rate within the hydrophobic phase. It is concluded that the lipid physical state probably does not affect the enzyme activity by altering the protein conformation but more likely does so by affecting the motion of the molecular participants in the reaction.     

Isolation of characterization of a major outer membrane protein of Pseudomonas aeruginosa. Evidence for the occurrence of a lipoprotein.

In the outer membrane of P. aeruginosa, a protein of apparent molecular weight 8,000 (protein I) is present as a major protein. Purification and chemical analysis of protein I were carried out. This protein was purified by essentially the same procedure as for the purification of the E. coli lipoprotein, which was developed by Inouye et al. (J. Bacteriol. (1976) 127, 555--563). The amino acid composition of protein I was determined. Protein I lacks proline, valine, isoleucine, phenylalanine, tryptophan, and half-cystine. Fatty acid analysis of the protein revealed that it contained 0.89 mol of fatty acids per mol of protein. Among the fatty acids hexadecanoic acid (C16:0) was predominant. In an in vivo labeling experiment, [2-3H]glycerol was incorporated into protein I. A protein with similar mobility to protein I on urea-SDS polyacrylamide gel electrophoresis was isolated from the purified peptidoglycan of P. aeruginosa by trypsin digestion. The amino acid composition of this protein was essentially the same as that of protein I. These results indicate that the outer membrane of P. aeruginosa contains a protein analogous to the E. coli lipoprotein, although considerable differences were observed in the amino acid composition and the fatty acid content.     

Bacteriophage T4 baseplate components. I. Binding and location of the folic acid.

Two different proteins with high affinities for the pteridine ring of folic acid have been used to determine the location of this portion of the folate molecule in the tail plate of T4D and other T-even bacteriophage particles. The two proteins used were (i) antibody specific for folic acid and (ii) the folate-binding protein from bovine milk. Both proteins were examined for their effect on various intact and incomplete phage particles. Intact T2H was weakly inactivated by the antiserum but not by the milk protein. No other intact T-even phage, including T4D, was affected by these two proteins. When incomplete T4D particles were exposed in an in vitro morphogenesis system, it was found that neither of the two proteins affected either the addition of the long tail fibers to fiberless particles or the addition of tail cores to tail plates. On the other hand, these two proteins specifically blocked the addition of T4D gene 11 product to the bottom of T4D baseplates. After the addition of the gene 11 protein, these two reagents did not inhibit the further addition of the gene 12 protein to the baseplate. It can be concluded that the phage folic acid is a tightly bound baseplate constituent and that the pteridine portion of the folic acid is largely covered by the gene 11 protein.     

Studies on the high-sulphur proteins of reduced mohair. The isolation and amino acid sequence of protein scmkb-m1.2.

The complete amino acid sequence of mohair protein, SCMKB-M1.2 (97 residues), was determined. The protein was isolated from reduced and carboxymethylated mohair by chromatography on DEAE-cellulose phosphate. Peptides for sequence determination were obtained by digestion with trypsin, pepsin, chymotrypsin, thermolysin and papain, and were fractionated by DEAE-cellulose chromatography, paper chromatography and electrophoresis. The sequence of the peptides were determined by the Edman degradation method (by use of both the Beckman Sequence and a non-automatic procedure), and by partial acid hydrolysis. The protein is closely homologous to wool protein SCMKB-IIIB2, and also contains acetylated alanine as N-terminal amino acid.     

Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.     

Protein kinases associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates.

When highly purified myelin from rat sciatic nerve was incubated with [gamma-32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [gamma-32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3',5'-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

Response of renal calcium-binding protein. Independence of kidney vitamin D hydroxylation.

Dietary calcium and dietary phosphorus restriction were studied in chicks fed either cholecalciferol or 1alpha-hydroxycholecalciferol. Intestinal calcium absorption and calcium-binding protein of 1alpha-hydroxycholecalciferol-treated chicks remained unchanged under dietary calcium restriction, but increased under dietary phosphorus restriction. Kidney calcium-binding protein was not altered by dietary caclium restriction in chidks treated with either cholecalciferol or 1alpha-hydroxycholecalciferol, but increased under dietary phosphorus restriction independent of the vitamin D source. In contrast to the intestine, calcium-binding activity of the kidney was found to be poorly related to the calcium-binding protein concentration. It is suggested that kidney calcium-binding protein is regulated by a mechanism different from that of intestinal calcium-binding protein, and that its concentration in renal tissue is related to renal caclium excretion or plasma calcium level.     

Calcium-binding protein of bovine intestine. The complete amino acid sequence.

The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.