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M Michalak

Publications and source records attributed to M Michalak.

120 records · Page 7Linked to original sources
PubMed1978

Studies on the heterogeneity of sarcoplasmic reticulum vesicles.

Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15--20% of total microsomes) and a heavy (80--85%) fraction by density gradient centifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca2+-binding proteins. (ii) The 105000 dalton protein of the light fraction cross-reacts immunologically with the Ca2+-dependent ATPase of the heavy fraction. (iii) Ca2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of "inside-out" vesicles by the following criteria: (i) No Ca2+ accumulation can be measured and the Ca2+-dependent ATPase activity is low and variable. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction. The light sarcoplasmic reticulum fraction has a higher phospholipid to protein ratio than the heavy one. This is consistent with the possibility that the two fractions derive from different parts of the sarcoplasmic reticulum system.

Adenosine Triphosphatases↗
PubMed1977

Sarcoplasmic reticulum vesicles and glycogen-protein particles in microsomal fraction of skeletal muscle.

1. Heavy microsomal fraction (HM) of rabbit skeletal muscle obtained by differential centrifugation between 8 000-30 000 g and consisting of sarcoplasmic reticulum (SR) vesicles contains variable amounts of glycogen and reveals some activity of phosphorylase b. The monomer of this enzyme of mol. wt. about 100 000 co-migrates in SDS-polyacrylamide gel electrophoresis with the main SR protein--Ca2+,Mg2+--dependent ATPase. 2. The highest specific activity of phosphorylase and the highest content of glycogen is present in the light microsomal (LM) fraction (30 000-100 000 g). 3. Contrary to the ATPase, phosphorylase b is released from the microsomal fraction by treatment with EDTA and is resistant to trypsin. 4. Both HM and LM fractions can be further fractionated on continuous sucrose density gradient at high speed. Main fraction of HM consists of highly purified SR vesicles. The second, small fraction of HM is identical with the main fraction of LM and consists of two populations: vesicles of structure and properties different from those of SR vesicles, and the particles of a complex of glycogen with some glycolytic enzymes.

Animals↗
PubMed1975

Changes in the structure, composition and function of sarcoplasmic-reticulum membrane during development.

The structure, chemical composition and function of the microsomal fraction, isolated by differential centrifugation and purified on sucrose gradients, from muscle of fetal, newborn and young rabbits were characterized and compared with those of sarcoplasmic reticulum vesicles from adult muscle. Negative staining shows that the microsomal vesicles isolated from muscles of embryos and newborn animals are smooth, in contrast to vesicles obtained from adult muscle which contain 4-nm particles on their surface. The particles appear first in the microsomal vesicles from muscles of 5--8-day-old rabbits. Their number increases with the age of the animals. Ca2+-pump protein, with molecular weight about 100000, accounts for 10% of the total protein content in sarcoplasmic reticulum membrane, isolated at the earliest stages of development analysed. Its amount increases continuously with the rabbit's age to the adult value of about 70% of total sarcoplasmic reticulum protein. The low amount of 100000-dalton protein and lack of 4-nm surface particles in sarcoplasmic reticulum vesicles obtained from fetal and newborn rabbits are strictly correlated with the low activity of Ca2+-dependent ATPase and the ability to take up Ca2+. These activities rise in parallel with the age of the rabbits. On the other hand, Mg2+-dependent ATPase activity is very high at the early stages of development and declines continuously to a low value in sarcoplasmic reticulum from adult muscle. The sarcoplasmic reticulum membrane from fetal and newborn rabbits contains a higher amount of lipids as compared with the membrane present in the muscle of adult animals. The ratio of both phospholipid to protein and neutral lipid to protein decreases with the age of the rabbits. The composition of sarcoplasmic reticulum phospholipids also changes during development.

Adenosine Triphosphatases↗
PubMedDate not supplied

Is cysteine residue important in FITC-sensitive ATP-binding site of P-type ATPases? A commentary to the state of the art.

Treatment of P-type ATPases (from mammalian sources) by fluorescein isothiocyanate (ITC) revealed the ITC label on a lysine residue that was than considered as essential for binding of ATP in the ATP-binding site of these enzymes. On the other hand, experiments with site directed mutagenesis excluded the presence of an essential Iysine residue that would be localized in the ATP binding sites of ATPases. Other previous studies, including those of ourselves, indicated that the primary site of isothiocyanate interaction may be the sulfhydryl group of a cysteine residue and this may be essential for binding of ATP. In addition considerable knowledge accumulated since yet also about the differences in stability of reaction product of isothiocyanates with SH- or NH2- groups. Based upon evaluation of the data available up to now, in present paper the following tentative roles for lysine and cysteine residues located in the ATP-binding site of P-type ATPases are proposed: The positively charged micro-domain of the lysine residue may probably attract the negatively charged phosphate moiety of the ATP molecule whereas the cysteine residue may probably be responsible for recognition and binding of ATP by creation of a proton bridge with the amino group in position 6 on the adenosine ring of ATP.

Adenosine Triphosphatases↗
PubMedDate not supplied

Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum.

The endoplasmic reticulum is a centrally located organelle which affects virtually every cellular function. Its unique luminal environment consists of Ca(2+) binding chaperones, which are involved in protein folding, post-translational modification, Ca(2+) storage and release, and lipid synthesis and metabolism. The environment within the lumen of the endoplasmic reticulum has profound effects on endoplasmic reticulum function and signaling, including apoptosis, stress responses, organogenesis, and transcriptional activity. Calreticulin, a major Ca(2+) binding (storage) chaperone in the endoplasmic reticulum, is a key component of the calreticulin/calnexin cycle which is responsible for the folding of newly synthesized proteins and glycoproteins and for quality control pathways in the endoplasmic reticulum. The function of calreticulin, calnexin and other endoplasmic reticulum proteins is affected by continuous fluctuations in the concentration of Ca(2+) in the endoplasmic reticulum. Thus, changes in Ca(2+) concentration may play a signaling role in the lumen of the endoplasmic reticulum as well as in the cytosol. Recent studies on calreticulin-deficient and transgenic mice have revealed that calreticulin and the endoplasmic reticulum may be upstream regulators in the Ca(2+)-dependent pathways that control cellular differentiation and/or organ development.

Animals↗
PubMedDate not supplied

Calcium storage in nonmuscle tissues: is the retina special?

It is now well established that calreticulin is a high capacity Ca(2+)-binding protein which is a major Ca2+ storage protein of the lumen of endoplasmic reticulum membranes in a wide variety of tissues with the exception of skeletal and cardiac muscles. However, in nervous tissue, confusion exists regarding the nature of the intracellular Ca2+ stores, as the organelle responsible for Ca2+ storage has been identified as the endoplasmic reticulum by some investigators and as the specialized organelle, calciosome by others. Calreticulin, calsequestrin, and calsequestrin-like proteins have all been, on different occasions, reported to be present in calciosomes. Cerebral and cerebellar tissues, moreover, have been shown to contain somewhat different systems of Ca(2+)-buffering proteins. In the present paper we discuss evidence that the Ca2+ storage systems of the retina may prove to be more complex than those of other neuronal tissues. Biochemical and immunocytochemical evidence indicates the presence of either an isoform of calreticulin or another protein that is antigenically similar to calreticulin, but of slightly higher molecular weight, in the endoplasmic reticulum of both neurons and Müller glia from rabbit neural retina. However, as retinal neurons express Purkinje cell markers, one may expect to observe the presence of calsequestrin in these cells as well. Secondly, antibodies against the onchocercal RAL-1 antigen recognize a protein sharing 62-65% amino acid sequence identity with calreticulin. The anti-RAL-1 antibodies show specificity for the retina. Whether or not the RAL-1 antigen is an active part of the Ca2+ storage systems of the retina remains to be verified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Calcium uptake during mitogenic stimulation of human lymphocytes: characterization of intracellular calcium compartments and demonstration of the presence of immunoreactive calrecticulin.

Phytohemagglutinin (PHA) stimulation of human peripheral blood lymphocytes (HPBL) rapidly increases 45Ca2+ uptake into intracellular pools. Detectable increase in 45Ca2+ uptake occurred only on exposure to mitogenic lectins but not with non-mitogenic lectins. However, intracellular free Ca2+ concentration [(Ca2+)i] increased comparably on exposure to either mitogenic or non-mitogenic lectins. Permeabilization of 45Ca2+ loaded cells revealed distinct pools of Ca2+ uptake. The highly digitonin sensitive pool #I (permeabilized by 0.02% digitonin) exchanged slowly and included a part that represented endoplasmic reticulum. Pool II was defined by lower digitonin sensitivity, had a much faster initial uptake. Pool III was digitonin-resistant and predominantly non-vesicular. During the first 120 min of PHA stimulation, significant increase in 45Ca2+ uptake occurred only into pool II. Progressive increase in uptake into pool I then occurred so that by 24 hours, this pool constituted the major fraction of PHA induced increment in total 45Ca2+ uptake. Using specific antibody to the calcium binding protein calreticulin, an analogous immunoreactive protein was detectable in resting HPBL. PHA stimulation led to a striking increase in abundance of immunoreactive calreticulin so that 24 hrs after PHA stimulation, there was a 28 and 3.4 fold increase in the amount of immunoreactive calreticulin present in the non-particulate fraction and the total particulate membrane fraction, respectively. A major part (72%) of the total cellular immunoreactive calreticulin in PHA stimulated cells at 24 hrs was released into the medium after permeabilization of lymphocytes with 0.02% digitonin, corresponding to the location of calcium uptake pool I.

Autoantigens↗
PubMedDate not supplied

Sarcoplasmic reticulum membrane and heart development.

Intracellular Ca2+ concentrations in cardiac cells are dependent on trans-sarcolemmal Ca2+ fluxes and the ability of sarcoplasmic reticulum to release and take up Ca2+. Ca2+ accumulation by sarcoplasmic reticulum membranes causes muscle to relax, whereas Ca2+ release from sarcoplasmic reticulum initiates contraction. Ca2+ transport by the sarcoplasmic is mediated by a Ca2+-dependent ATPase enzyme. Ca2+ release from sarcoplasmic reticulum may be mediated by a ligant-gated Ca2+ channel. The physiological role of sarcoplasmic reticulum in developing muscle is not well established. In this report we investigated the composition and function of sarcoplasmic reticulum membranes during cardiac myogenesis. Phospholamban, a major phosphoprotein in mature sarcoplasmic reticulum membranes was present during early stages of cardiac myogenesis. The embryonic form of phospholamban was phosphorylated by cAMP-dependent protein kinase but not in the presence of Ca2+ and calmodulin. Ca2+ uptake and Ca2+-dependent ATPase activity were low in fetal sarcoplasmic reticulum compared to adult control membranes, although the apparent affinities of the enzyme for Ca2+ were similar. Sarcoplasmic reticulum vesicles used in these studies had very low levels of plasma membrane and mitochondrial contamination. The amounts of both 110-kDa Ca2+-ATPase and 55-kDa calsequestrin in the sarcoplasmic reticulum membrane were lower in fetal sarcoplasmic reticulum vesicles compared to mature membranes. Ca2+-ATPase and calsequestrin were identified in the isolated sarcoplasmic reticulum vesicles using specific antibodies produced against these membrane proteins. Age-related differences in Ca2+ transport properties of cardiac sarcoplasmic reticulum and in the amount of Ca2+-ATPase and calsequestrin may explain alterations in the regulation of intracellular Ca2+ concentrations in fetal heart muscle. This may relate to the developmental changes observed in myocardial function.

Adenosine Triphosphatases↗