Calmodulin an activator of human erythrocyte (Ca2+ + Mg2+)-ATPase phosphorylation

The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130 000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca²⁺ + Mg²⁺)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca²⁺ + Mg²⁺)-ATPase of human erythrocyte membranes.     

Some properties of the purified (Ca2+ + Mg2+-ATPase from human red cell membranes

The (Ca²⁺ + Mg²⁺-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tej?ka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81–88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two K_m values of 3.5 and 120 μM. The enzyme is phosphorylated by ATP in the presence of Ca²⁺ only.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH₂:cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands.

2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c₁, band 5 the Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c₁ and identified with the so-called oxidation factor, and band 7 a polypeptide associated with cytochrome b.

3. The validity of molecular weight estimates for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively.

4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide bands.     

Purification and characterization of glutathione disulfide-stimulated Mg2+-ATPase from human erythrocytes

We have previously shown the presence of two different forms of glutathione disulfide (GSSG)-stimulated Mg2+-ATPases in human erythrocytes. We have now investigated a low-Km form of the enzyme from human erythrocytes. Purification of the enzyme was performed to apparent homogeneity involving procedures of affinity chromatography and gel filtration. The enzyme was composed of two non-identical subunits of Mr = 82K and 62K. The enzyme reconstituted into phospholipid vesicles showed both GSSG-stimulated Mg2+-ATPase activity (285 nmol Pi released/mg protein/min) and active GSSG transport activity (320 nmol GSSG/mg protein/min). The amino acid composition of the enzyme was similar to that of the enzyme purified from cytoplasmic membranes of human hepatocytes. These enzymes were immunologically cross reactive. These results indicate that this enzyme functions in the active transport of GSSG as it possibly does in hepatocytes.     

Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and golgi membranes from rat liver

Rough and smooth microsomes and Golgi membranes incorporate $\text{N-}\text{acetylglucosamine}$ from $\text{UDP}\text{-N-}\text{acetylglucosamine}$ into endogenous protein acceptors. A lipid intermediate of the dolichol phosphate type participates in this transfer reaction in the case of both microsomal subfractions, but the nature of lipid glycosylation is different in these two fractions. Glucosamine transfer in Golgi membranes does not appear to involve a lipid intermediate. In contrast to the results obtained under in vivo conditions, no glucosamine label is recovered in nascent ribosomal proteins or on luminal secretory proteins after incubation in vitro. Proteolysis of intact vesicles of the subfractions removes glycosylated dolichol phosphate and protein acceptors to various extents and interferes with transferase activities. This finding suggests the possibility that glycosylation at the cytoplasmic side of the membrane of the endoplasmic reticulum may involve a system separate from that acting at the luminal side of the same membrane.     

Structural and functional degradation of Ca2+:Mg2+-ATPase rich sarcoplasmic reticulum vesicles photosensitized by erythrosin B

Erythrosin B (Red Dye No. 3) and Rose Bengal photosensitize the destruction of the Ca2+:Mg2+-ATPase pump protein in sarcoplasmic reticulum (SR) vesicles with respective quantum efficiencies of (1.53 +/- 0.19) X 10(-3) and (1.25 +/- 0.18) X 10(-3). Damage to vesicle function was assayed by measurements of increases in passive Ca2+ permeability. Rates of passive Ca2+ movement into the SR lumen were increased by dye photosensitization in proportion to radiation absorbed. Active Ca2+ transport into SR vesicles was blocked independent of radiation absorbed by Erythrosin B and Rose Bengal at free concentrations of 0.69 microM and 1.16 microM, respectively. The photochemical lability of the Ca2+ pump protein and alterations in passive and active Ca2+ transport may be dependent on the concentration of the dye in the membrane. The photosensitization results may have implications with respect to the suitability of Erythrosin B usage in vivo, since the brightness of our irradiation source is comparable to that of sunlight at 480 nm.     

A high molecular weight platelet aggregating factor in Crocus sativus

Bulbs of Crocus sativus, variety Cartwrightianus contain a protein factor with aggregating properties on human platelets. This factor was purified by different chromatographic techniques and shows a molecular weight of 42 000, as it was estimated by Sephadex G-75 column chromatography and sodium dodecyl sulfate (SDS) polyacrylamide slab gel electrophoresis.     

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides

The pigment content of a B800–850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed.The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800–850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band.The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.     

Conditionally lethal mutants of bacteriophage T4 defective in production of a transfer RNA

The two buried carboxyls (Asp-102 and Asp-194) in both chymotrypsin and chymotrypsinogen are ionized at pH values greater than 4.2 and may be ionized even as low as pH 3.This was demonstrated by coupling most of the surface carboxyis of the proteins by a carbodi-imide with glycinamide or semicarbazide to diminish the groups ionizing at low pH and then titrating the proton uptake on denaturation by sodium dodecyl sulphate between pH 3.0 and 4.6. At pH values greater than 4.2 all unblocked carboxyls are ionized. The proton uptake during the conformational change on denaturation was determined by a stopped-flow procedure and found to be about 2H⁺/mol between pH 3.0 and 3.6. The rate constant for the uptake of protons is the same as that for the exposure of tryptophan and lies in the tens of millisecond region.The buried negative charge at the active site appears to be mainly on Asp-102 rather than on His-57, the pK_a of which must be raised by the buried charge. This enhances its efficacy as a base catalyst in the “charge relay system”.The presence of an intact charge relay system in the inactive zymogen illustrates the importance of stereochemical fit between enzyme and substrate. Enzyme catalysis could hardly be mediated by a catalyst which is uniquely reactive in the absence of correct enzyme-substrate orientation as this would be inconsistent with its specificity.     

Gene 68, a new bacteriophage T4 gene which codes for the 17K prohead core protein is involved in head size determination

We have identified the gene for a major component of the prohead core of bacteriophage T4, the 17K protein. The gene, which we call gene 68, lies between genes 67 and 21 in the major cluster of T4 head genes. All of the genes in this region of the T4 genome have overlapping initiation and termination codons with the sequence T-A-A-T-G. We present the DNA sequence of the gene and show that it codes for a protein containing 141 amino acids with an acidic amino-terminal half and a basic carboxyl terminus. Antibodies prepared against the 17K protein were used to show that it is cleaved by the phage-coded gp21 protease during head maturation and that most of the protein leaves the head after cleavage. A frameshift mutation of the gene was constructed in vitro and recombined back into the phage genome. The mutated phages had a drastically reduced burst size and about half of the particles produced were morphologically abnormal, having isometric rather than prolate heads. Thus, the 17K protein is involved in head shape determination but is only semi-essential for T4 growth.     

Structural and functional properties of chromatophores and membrane vesicles from Rhodepseudomonas sphaeroides

Membrane vesicles have been isolated by a modified procedure from Rhodopseudomonas sphaeroides, grown phototrophically under high light intensity. In addition,chromatophores have been isolated from this organism grown phototrophically with low light intensities.Structural, chemical and functional properties of both preparations have been investigated and compared. The orientation of the membrane preparations has been studied by freeze-etch electron microscopy, the localization of cytochrome c₂, and light-driven active transport of amino acids and Ca²⁺. The results demonstrate that the orientation of the vesicle membrane is the same as the cytoplasmic membrane of intact cells; the membranes in chromatophores, however, have an inverted orientation.On a dry weight basis, the membrane vesicles contain less protein, carotenoids and bacteriochlorophyll and more lipids than do chromatophores. Qualitatively, however, the composition of both preparations is similar.It is concluded that the intracytoplasmic structures from which the chromatophores are derived are structurally and functionally similar to (and most likely continuous with) the cytoplasmic membranes from which the vesicles are derived.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.     

Exploring structural homology of proteins.

A method for systematically comparing the folding of the three-dimensional structures of proteins has been developed. A search function, plotted in terms of three Eulerian angles, represents the number of sequentially equivalenced amino acids. For each orientation one protein structure is rotated about its center of mass with respect to the other and probabilities are calculated which estimate the degree of structural parallelism. The structurally equivalent residues with highest probabilities are then selected for the best common topology. It was observed that, when structures containing about 150 residues were compared, the random background had a mean value of around 14 residues and the standard deviation was approximately nine residues. The method has been shown to be successful in determining the similarity of the NAD binding domains of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, and in comparing the heme binding fold of cytochrome b₅ with the globins.Application of the method to compare hen egg white lysozyme and T4 phage lysozyme led to a single significant peak of 62 residues. The structural homology indicated by this peak showed that the substrate, as bound to hen egg white lysozyme, has a corresponding binding site in the large cleft of the phage lysozyme. The predicted binding site of N-acetyl glucosamine at position C compares well with an N-acetyl glucosamine center observed to bind to crystalline phage lysozyme (B. W. Matthews, personal communication).Some results for the comparison of the two Fe-S cage binding domains of ferredoxin are also presented.