Obesity Paradox in Caucasian Seniors: Results of the PolSenior Study.

OBJECTIVES: To investigate the influence of overweight and obesity on general performance and mortality in seniors. DESIGN: Cross-sectional multidisciplinary study on ageing of the Polish population. SETTING: Community-dwelling individuals aged 65 years or older, selected using three-stage stratified, proportional draw. PARTICIPANTS: 4944 Polish Caucasian seniors, aged 65 years or older recruited between October 2007 and October 2010. MEASUREMENTS: All study subjects underwent measurement of body mass index (BMI), waist circumference (WC), and arm circumference (AC). The physical and cognitive performance was evaluated using the Katz Activities of Daily Living (ADL) score and Mini-Mental State Examination (MMSE), respectively. Morbidity data were obtained from a medical questionnaire. Mortality data were obtained from the Population Register of Poland between October 2015 and October 2018. RESULTS: Increasing age was associated with a decreased prevalence of obesity (all p<0.001). Higher BMI, WC and AC values were associated with higher ADL and MMSE scores (all p<0.001). On multivariate analysis, all three body measurements in women remained independent predictors of the ADL score (BMI p=0.002, WC p=0.005, AC p<0.001) and MMSE score (p<0.001, p=0.003, p<0.001). In men, physical functioning was associated with AC (p=0.003), and cognitive status was associated with AC (p<0.001) and BMI (p=0.013). There was no association between general obesity, abdominal obesity, or AC with several aging-related adverse conditions. Kaplan-Meier survival curves showed that overweight and obesity were associated with the lowest mortality. On multivariate analysis, BMI and AC values remained independent predictors of mortality. In successfully aging individuals, neither BMI, WC, nor AC remained such predictors. CONCLUSIONS: Overweight and obesity in Caucasian seniors are not associated with deterioration of physical and cognitive function or with increased mortality.

Atrial fibrillation independently linked with depression in community-dwelling older population. Results from the nationwide PolSenior project.

BACKGROUND: Depression is a frequently observed comorbid condition in patients with cardiovascular diseases. In contrast to coronary heart disease and heart failure there is a limited amount of published data concerning the increased prevalence of depression among patients with atrial fibrillation (AF). Therefore, we decided to assess the prevalence of depression in Polish community-dwelling older patients with a history of AF. METHODS: The data were collected as part of the nationwide PolSenior project (2007-2012). Out of 4979 individuals (age range 65-104 years), data on self-reported history of AF were available for 4677 (93.9%). Finally, 4049 participants without suspected moderate or severe dementia in Mini Mental State Examination test were assessed with the 15-item Geriatric Depression Scale (GDS), and a score of 6 points and more was regarded as suspected depression. RESULTS: Mean age (±SD) of the study population was 78.1 (±8.3) years; 52% were males. The history of AF was reported by 788 (19.5%) subjects. In the univariate analysis a self-reported AF history was associated with 42% increase of suspected depression (41% vs 29%; P < 0.001). In multivariate logistic regression AF remained an independent predictor of depression (OR = 1.69; 95%CI: 1.43-2.00), stronger than heart failure, diabetes or coronary heart disease. CONCLUSIONS: In community-dwelling geriatric Polish population AF is associated with higher prevalence of depression. This association is independent from the demographic factors, disabilities and comorbidities (including history of stroke).

Prevalence of anemia in relation to socio-economic factors in elderly Polish population: the results of PolSenior study.

Anemia is an independent risk factor for functional decline and mortality among older adults. Since mild anemia in older people is often under-diagnosed and ignored, its prevalence needs precise determination and recognition of predisposing factors. None of the previous studies based on the data obtained from the representative elderly population identified the influence of socio-economic factors on the prevalence of anemia. PolSenior was a cross-sectional population-based study performed on the nationally representative sample of Polish seniors. Complete blood count was assessed in 4003 respondents aged 65 years or above (1910 women) divided into six five-year cohorts and a reference group of 622 people aged 55 - 59 years (333 women). Anemia was defined based on the WHO criteria: Hb < 12.0 g/dL in women and Hb < 13.0 g/dL in men. The following socio-economic factors were evaluated through the multiple logistic regression analysis: education level, marital status, place of residence, living arrangements and self-reported poverty. The prevalence of anemia in older persons standardized for the population was 10.8% (17.4% of the study group) and was more frequent in men than in women (20.8% versus 13.6%). The frequency of anemia progressed with age from 5.3% in the youngest to 37.7% in the oldest cohort, and the progression was higher in men. The multiple logistic regression analysis revealed the link between anemia and age in both genders, as well as unmarried status and urban dwelling in men. When age was not taken into account, logistic regression showed the link between anemia and unmarried status, urban place of residence (both genders), and low level of education (women only). Among seniors, those poorly educated, unmarried and city inhabitants require intense screening for anemia.

Health status correlates of malnutrition in the polish elderly population - Results of the Polsenior Study.

OBJECTIVE: The aim of the study was to analyze health-related factors associated with poor nutritional status (PNS) of a representative group of Polish older people, based on data from the PolSenior project (the first nation-wide study of Polish senior citizens). PATIENTS AND METHODS: Nutritional status was assessed in 3751 community-dwelling older people (1770 females, mean age: 77.4±8.0 years) using the Mini Nutritional Assessment - Short Form. Elements of comprehensive geriatric assessment (cognitive and mood screening), selected medical data were analyzed in relation to the nutritional status. These were: the number of medications, the number of chronic diseases, selected diseases potentially related to malnutrition (anaemia, stroke, peptic ulcer, Parkinson's disease, cancer - past or present), total edentulism, use of dentures, and chronic pain. RESULTS: PNS was observed in 44.2% of participants. Female sex [OR 1.72, 95% Cl (1.45-2.04)], advanced age [OR 2.16 (1.80-2.58)], symptoms of depression [OR 11.52 (9.24-14.38)], cognitive impairment [OR 1.52 (1.20-1.93], multimorbidity [OR 1.27 (1.04-1.57)], anaemia [OR 1.80 (1.41-2.29)] and total edentulism [OR 1.26 (1.06-1.49)] were independently correlated with PNS. CONCLUSIONS: PNS in Polish elderly population is strongly related to the occurrence of symptoms of depression. People in advanced age with symptoms of depression, cognitive impairment, multimorbidity, anaemia and total edentulism should be screened and monitored for early symptoms of malnutrition.

Social and economic correlates of malnutrition in Polish elderly population: the results of PolSenior study.

OBJECTIVES: The aim of this study was to evaluate the prevalence of malnutrition in Polish elderly population and analyse its social and economic correlates based on the data from the PolSenior project, the first large-scale study of a representative group of Polish seniors. DESIGN: A cross-sectional population-based study. SETTING: All territorial provinces in Poland. PARTICIPANTS: 4482 community-dwelling respondents aged 65 years or above (women: n=2142, age=79.0±8.4 years; men: n=2340, age= 78.3±8.6 years). MEASUREMENTS: The nutritional status of participants was assessed through the Mini Nutritional Assessment Short Form (the revised MNA-SF). Out of social and economic correlates we evaluated age, sex, level of education, marital status, place of residence, living conditions and economic status. Economic status of the respondents was determined on the basis of questions on how well they could manage their own budgets. Those who could afford only the cheapest food or clothes were considered the group of self-reported poverty. RESULTS: Frequency of malnutrition in the PolSenior population accounted for 7.5% (in 5.0% men and 9.0% women; p<0.001). The risk of malnutrition was present in 38.9% (33.3% men and 42.4% women; p<0.001). In our study female sex, older age, unmarried status, living in a rural area and self-reported poverty were independent correlates of malnutrition. CONCLUSIONS: Our data showed high prevalence of malnutrition and the risk of its development among the community-dwelling elderly people in Poland. Screening with MNA-SF should focus in particular on unmarried, poorly educated individuals, in late old age, living in rural areas and self-reporting a poor financial state, especially women.

Plasma level of N-terminal pro brain natriuretic peptide (NT-proBNP) in elderly population in Poland--the PolSenior Study.

BACKGROUND: The brain natriuretic peptides (BNP, NT-proBNP) are useful diagnostic markers of heart failure (HF), as exemplified by the ESC Heart Failure guidelines. The PolSenior project was an epidemiological study carried out to examine medical, psychological and socioeconomic aspects of aging in Poland. The aim of this study is an epidemiological description of HF based on elderly population from the PolSenior Study, stratified by NT-pro-BNP concentration values. MATERIAL AND METHODS: The research sample included 4979 respondents (2567 males and 2412 females) split into six equally sized age groups of elderly individuals. The study consisted of three visits performed by trained nurses and included a questionnaire survey, comprehensive geriatric assessment and blood and urine sampling with more than 50 biochemical parameters measured. Serum NT-pro-BNP was measured by electrochemiluminescence method (ECLIA). RESULTS: The prevalence of chronic kidney disease (CKD) (77.8%) and atrial fibrillation (39.5%), number of hospitalizations (23.7%) and number of patients treated with HF drugs were highest in NT-proBNP > 2000 pg/ml group and least frequent in NT-proBNP < 400 pg/ml group. Obese patients had significantly more frequently NT-proBNP values < 400 pg/ml (73.0%) and less frequently NT-proBNP values >2000 pg/ml (2.8%). Age over 70 years and male gender were associated with the increased NT-pro-BNP (> 400 pg/ml) (OR 1.41; CI 1.20-1.65 for male gender). CONCLUSIONS: We conclude that CKD and atrial fibrillation are associated with the occurrence of increased NT-pro-BNP, the surrogate for HF in elderly population. On the contrary, overweight or obesity is associated with lower prevalence of HF in elderly.

Drug consumption among Polish centenarians.

The aim of the study was to describe the quantitative and qualitative aspects of pharmacotherapy of Polish centenarians. The studied group consisted of 92 centenarians (mean age: 101.7±1.2 years, 77 females, mean age: 101.5±1.2; 15 males mean age: 102.2±1.2). Among the studied subjects, 18 individuals (19.6% of all subjects) did not use any drugs in his or her daily regimen. The mean number of drugs per person was 2.5±2.5 drugs (prescription drugs: 1.9±2.2 and non-prescription drugs: 0.5±0.8). Fifty-six centenarians (60.9% of all studied subjects) took concomitantly 0-3 drugs daily while 36 (39.1%) took more than 3 drugs daily. Within this group, 30 centenarians (32.6%) took 5 or more drugs concomitantly every day. The most commonly used groups of drugs were: gastrointestinal drugs (55 centenarians, 74.3% of all drug consumed), cardiovascular drugs (51 centenarians, 68.9%) and central nervous system drugs (N) (38 centenarians, 51.4%). In the studied group, 6 persons (8.1% of all drug consumers) were taking one potentially inappropriate drug based on the Beers criteria. To conclude, the mean number of drugs, the prevalence of polypharmacy, and the tendency for potential inappropriateness of treatment are lower among Polish centenarians comparing to the common elderly.

An age-related decrease in factor V Leiden frequency among Polish subjects.

Factor V Leiden (G1691A FV mutation) is a widely acknowledged risk factor of deep vein thrombosis, including pulmonary embolism as the most serious complication. However, its high prevalence of ~5%in the Caucasian population might be related to an unknown evolutionary advantage. It might exert a beneficial effect on the carrier, e.g. protecting women from excessive bleeding during labour or allowing increased survival in severe sepsis or with other inflammatory diseases. The aim of our study was to verify or contradict the hypothesis of a favourable association between the A allele (A1691) and longevity in the Polish population. For this purpose, the G1691A mutation was analyzed by PCR-RFLP in 1016 Poles: 400 neonates (187 female and 312 male), 184 healthy adults (129 female and 55 male), and 432 long-lived individuals (age ≥95 years: 343 women and 89 men). Frequencies of G1691A carriers and the A1691 allele in long-lived individuals (0.2% and 0.1%, respectively) were significantly lower than in neonates (4.2% and 2.2%, respectively) and adults (3.3% and 1.6%). The frequency of the G1691A factor V Leiden mutation decreased with age, which indicates a shorter survival time among A1691 allele carriers in the Polish population.

Asthma, allergy, and respiratory symptoms in centenarians living in Poland.

According to National Census, there were 1541 people over the age of 100 years (centenarians) in Poland, in 2002, including 1215 females and 326 males. The aim of the present study was to assess the prevalence of asthma, allergy, and respiratory symptoms in centenarians included in the Polish Centenarians Program, POLSTU 2001, conducted between 2001 and 2004. The study group consisted of 301 subjects including 258 females and 43 males. Research data were gathered in a questionnaire designed exclusively for the study, completed by the interviewer during meetings with the subjects and their families. According to the medical history reported by the subjects, 10 persons (3.3%) suffered from asthma and 41 (13.6%) from allergy. There were no subjects with childhood onset asthma. Allergy to food and medicinal products was the most prevalent. One in four centenarians reported dyspnea and one in eight complained of cough. When analyzed in relation to gender, cough was more prevalent in males, which might have been related to cigarette smoking. Respiratory disorders are frequent in elderly populations, but symptoms may be underreported, especially in the situation of coexisting medical problems. Moreover, it might be difficult to perform full diagnostic procedures in the very elderly due to disability, cognitive impairment, and technical problems. Thus, medical care for the aged should be based on thorough medical evaluation supported by the medical history and reliable information on physician-diagnosed diseases.

Anthropometric chest structure of Polish centenarians.

The aim of the study was to analyze somatometric chest structure in persons aged over 100 years. The study group included 83 women and 13 men, aged 100-108 yr (median age -100.8 yr), who participated in the scientific project: 'Genetic and Environmental Factors of Longevity of Polish Centenarians' in 2002-2004. The Rohrer index of chest structure using acromion-acromion length and body height were compared with the results of pulse oximetry, spirometry, and the level of general physical activity. The majority of the centenarians had a pyknic structure of the chest, most likely as a result of a progressive reduction of body height and chest stooping. In comparison with the women who had marked alterations of chest structure, females with less profound changes had a lower respiratory rate, better tolerance of exercise, higher forced vital capacity, and a higher physical activity. A small number of male subjects studied made it impossible to analyze statistical correlations in this group. We conclude that there is a need to redefine anthropometric indices for a reliable assessment of chest structure in senescent subjects.

Blood pressure in centenarians in Poland.

The objective of the study is assessment of the prevalence and type of hypertension in centenarians in Poland. The investigations included 92 people who had turned 100 years of age, who, within the protocol of the Project of Investigation Polish Centenarians, underwent genetic, anthropometric, psychological and sociological examinations, and whose cardiovascular system was assessed. In the present analysis, we are analysing data concerning their blood pressure (BP) assessed by several measurements (3-6) with the mercury sphygmomanometer on both arms in sitting (if possible) or lying position performed during one visit. Hypertension was diagnosed when average BP value exceeded > or = 160/95 or > or = 140/90 mmHg. The average of age was 101.2 years (range 100-111 years), the respective values for BP were: systolic 146.7 mmHg (99-213 mmHg), diastolic BP--80.3 mmHg (55-114 mmHg) and pulse pressure (PP) 66.4 mmHg (31-129 mmHg). Hypertension diagnosed based on the criterion > or = 160/95 mmHg was found in 29% of subjects, and according to the recent WHO criterion (> or = 140/90 mmHg) in 65% of subjects. PP exceeded 65 mmHg in 44.6%, and was above 50 mmHg in 91% subjects. In conclusion, hypertension occurs less frequently in centenarians, than in the entire population of old people, but it nevertheless cannot be considered a rare condition.

Kidney function estimated with different formulas in centenarians.

PURPOSE: There are growing doubts about the accuracy of Cockcroft-Gault formula (CG) used for the estimation of creatinine clearance, especially in elderly. Recently, the authors of the multicenter trial of the Modification of Diet in Renal Diseases (MDRD) have proposed a new equation. Moreover, Baracskay et al. (B), proposed the special formula for the estimation of kidney function (KF) in elderly. The aim of our study was to compare the results of KF calculated with these three formulas in centenarians. MATERIAL AND METHODS: The study involved 50 centenarian subjects aged 100-111 years (41 females and 9 males) who participated in Polish Centenarians Program. In all of them KF was estimated with the CG, B and MDRD formulas. RESULTS: In the whole population examined, the mean KF according to CG was lower in comparison to both others (p < 0.001 vs both B and MDRD). Also, in females CG results were the lowest (p < 0.001 vs both B and MDRD). In contrast, KF calculated according to CG and B did not differ in males. The results of the MDRD formula significantly exceeded the two others also in males (p < 0.001 vs CG and B). No impact of gender on the obtained results could be found when CG and MDRD were used. However, according to B, the mean values for females were higher (p < 0.01). CONCLUSIONS: KF calculated with the CG, B and MDRD formulas significantly differed in the centenarians examined. Thus, further studies, which include a reference standard, are necessary to answer the question which of these mathematical formulas is the most reliable for the calculation of KF in the elderly.

Actin-binding proteins of invasive malaria parasites and the regulation of actin polymerization by a complex of 32/34-kDa proteins associated with heat shock protein 70kDa.

Movement of the malaria parasite into a host erythrocyte during invasion is thought to involve polymerization of parasite actin. We have used F-actin affinity chromatography to isolate actin-binding proteins from Plasmodium knowlesi merozoites, in an attempt to identify proteins responsible for regulating parasite actin polymerization during invasion. Five major proteins, of molecular masses 75, 70, 48, 40 and 34 kDa, were reproducibly eluted from the F-actin columns. The 70 kDa actin-binding protein was identified by tryptic peptide microsequencing as heat shock protein-70 kDa (HSC70); this identification was confirmed by Western blotting with anti-HSC70 antibody, and binding of the protein to ATP-agarose. A doublet of 32/34-kDa proteins coeluted with parasite HSC70 from the F-actin and ATP-agarose columns; a complex of these three proteins was also observed by gel filtration chromatography Highly enriched fractions containing the Plasmodium HSC70/32/34 complex inhibited the polymerization of rabbit skeletal muscle actin, in vitro. This capping activity was calcium-independent, and abrogated by phosphatidylinositol 4,5-bisphosphate. The average length of the actin filaments polymerized in presence of the HSC70/32/34-kDa complex was significantly shorter than in the absence of the complex, consistent with a capping activity. The capping or uncapping of actin filament ends by the HSC70/32/34-kDa complex during invasion could provide a mechanism for localized actin filament growth and movement of the parasite into the host cell.

Ca2+ bound to the high affinity divalent cation-binding site of actin enhances actophorin-induced depolymerization of muscle F-actin but inhibits actophorin-induced depolymerization of Acanthamoeba F-actin.

The cation tightly bound to actin, Mg2+ or Ca2+, affects the ability of actophorin to accelerate depolymerization of filaments and bind to monomers of actin prepared from rabbit skeletal muscle and Acanthamoeba castellanii. Actophorin interacted similarly with muscle and Acanthamoeba Mg2(+)-F-actin but depolymerized muscle Mg2(+)-F-actin more efficiently. Muscle Ca2(+)-F-actin depolymerized about 5 times more rapidly than Mg2(+)-F-actin in the presence of actophorin but Acanthamoeba Ca2(+)-F-actin was highly resistant to actophorin. Muscle actin subunits dissociated more rapidly than Acanthamoeba actin subunits from copolymers of muscle and Acanthamoeba Ca2(+)-actin upon addition of actophorin although Acanthamoeba actin dissociated much more rapidly from copolymers than from its homopolymer. The Kd of the 1:1 complex between actophorin and monomeric actin was somewhat lower for muscle Mg2(+)-ATP-G-actin than for both Acanthamoeba Mg2(+)-ATP-G-actin and muscle Ca2(+)-ATP-G-actin. The data for the interactions of actophorin with Acanthamoeba Ca2(+)-ATP-G-actin or muscle and amoeba Mg2(+)- and Ca2(+)-ADP-G-actin were incompatible with the formation of 1:1 actin: actophorin complexes and, thus, Kd values could not be calculated. While it may not be surprising that actophorin would interact differently with Mg2(+)- and Ca2(+)-actin, it is unexpected that the nature of the tightly bound cation would have such dramatically opposite effects on the ability of actophorin to depolymerize muscle and Acanthamoeba F-actin. Differential severing by actophorin, with Acanthamoeba Ca2(+)-actin being almost totally resistant, is sufficient to explain the results but other possibilities cannot be ruled out.

Long-range conformational effects of proteolytic removal of the last three residues of actin.

Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process.

Proteolytic removal of three C-terminal residues of actin alters the monomer-monomer interactions.

Homogeneous preparations of actin devoid of the three C-terminal residues were obtained by digestion of G-actin with trypsin after blocking proteolysis at other sites by substitution of Mg2+ for the tightly bound Ca2+. Removal of the C-terminal residues resulted in the following: an enhancement of the Mg(2+)-induced hydrolysis of ATP in low-ionic-strength solutions of actin; an increase in the critical concentration for polymerization; a decrease in the initial rate of polymerization; and an enhancement of the steady-state exchange of subunits in the polymer. Electron microscopy indicated an increased fragility of the filaments assembled from truncated actin. The results suggest that removal of the C-terminal residues increases the rate constants for monomer dissociation from the polymer ends and from the oligomeric species.

Localization of the tightly bound divalent-cation-dependent and nucleotide-dependent conformation changes in G-actin using limited proteolytic digestion.

Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place. Replacement of the tightly bound Ca2+ by Mg2+ in ATP-actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues. It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C-terminal segment. Conversion of ATP-actin to ADP-actin (with Mg2+ as the tightly bound cation) abolished the protective effect of Mg2+ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39-51. We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule. These experiments demonstrated structural relationships between loop 39-51 and regions involving Lys61 and Lys68. It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer.

An EPR study of the rotational dynamics of actins from striated and smooth muscle and their complexes with heavy meromyosin.

The rotational motions of the actin from rabbit skeletal muscle and from chicken gizzard smooth muscle were measured by conventional and saturation transfer electron paramagnetic resonance (EPR) spectroscopy using maleimide spin-label rigidly bound at Cys-374. The conventional EPR spectra indicate a slight difference in the polarity of the environment of the label and in the rotational mobility of the monomeric gizzard actin compared to its skeletal muscle counterpart. These differences disappear upon polymerization. The EPR spectra of the two actins in their F form and in their complexes with heavy meromyosin (HMM) did not reveal any difference in the rotational dynamic properties that might be correlated with the known differences in the activation of myosin ATPase activity by smooth and skeletal muscle actin. Our results agree with earlier EPR studies on skeletal muscle actin in showing that polymerization stops the nanosecond rotational motion of actin monomers and that F-actin undergoes rotational motion having an effective correlation time of the order of 0.1 ms. However, our measurements show that complete elimination of the nanosecond motions requires prolonged incubation of F-actin, suggesting that the slow formation of interfilamental cross-links in concentrated F-actin solutions contributes to this process. We have also used the EPR spectroscopy to study the interaction between HMM and actin in the F and G form. Our results show that in the absence of salt one HMM molecule can cooperatively interact with eight monomers to produce a polymer which closely resembles F-actin in its rotational mobility but differs from the complex of F-actin with HMM. The results indicate that salt is necessary for further slowing down, in a cooperative manner, the sub-millisecond internal motion in actin polymer and for a non-cooperative change in the intramonomer conformation around Cys-374 on the binding of HMM.

Ca2+-calmodulin-dependent polymerization of actin by myelin basic protein.

The interaction between myelin basic protein (MBP) and G-actin was studied under nonpolymerizing conditions, i.e.,2mM HEPES, pH 7.5, 0.1 mM CaCl2 and 0.2 mM ATP. Fluorescence studies using pyrenyl-actin and the measurements of ATP hydrolysis rate show that MBP induces changes in the structure of the actin monomer similar to those occurring during polymerization by salt. Electron microscope observations of the MBP-G-actin complex reveal the presence of filamentous structures which appear as separate filaments or as bundles of filaments in lateral association. These filaments are polar as visualized by attachment of heavy meromyosin. The biochemical data together with electron microscope observations suggest that the binding of MBP to G-actin under non-polymerizing conditions induces an interaction between actin monomers leading to the formation of filamentous structures which may be similar to F-actin filaments. The effects of MBP on G-actin can be reversed by calmodulin in the presence of Ca2+.

Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin.

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.