Synthetic peptide of the sequence 632-642 on myosin subfragment 1 inhibits actomyosin ATPase activity.

A synthetic peptide corresponding to a sequence 632-642 (S632-642) on the myosin subfragment 1 (S-1) heavy chain and spanning the 50/20 kDa junction of S-1 binds to actin in the presence and absence of S-1. The binding of 1.0 mole of peptide per actin causes almost complete inhibition of actomyosin ATPase activity and only partial inhibition of S-1 binding to actin. The binding of S632-642 to the N-terminal segment of actin is supported by competitive carbodiimide cross-linking of S-1 and S632-642 to actin and the catalytic properties of cross-linked acto-S-1 and actin-peptide complexes. These results show that the sequence 632-642 on S-1 is an autonomous binding site for actin and confirm the catalytic importance of its interactions with the N-terminal segment of actin.     

Protein transfer between A-I-containing lipoprotein subpopulations: evidence of non-transferable A-I in particles with A-II.

Transfer of apolipoproteins (apo) between the two subpopulations of apo A-I-containing lipoproteins in human plasma: those with A-II [Lp(AI w AII)] and those without [Lp(AI w/o AII)], were studied by observing the transfer of 125I-apo from a radiolabeled subpopulation to an unlabeled subpopulation in vitro. When Lp(AI w AII) was directly radioiodinated, 50.3 +/- 7.4 and 19.5 +/- 7.7% (n = 6) of the total radioactivity was associated with A-I and A-II, respectively. In radioiodinated Lp(AI w/o AII), 71.5 +/- 6.8% (n = 6) of the total radioactivity was A-I-associated. Time-course studies showed that, while some radiolabeled proteins transferred from one population of HDL particles to another within minutes, at least several hours were necessary for transfer to approach equilibrium. Incubation of the subpopulations at equal A-I mass resulted in the transfer of 51.8 +/- 5.0% (n = 4) of total radioactivity from [125I]Lp(AI w/o AII) to Lp(AI w AII) at 37 degrees C in 24 h. The specific activity (S.A.) of A-I in the two subpopulations after incubation was nearly identical. Under similar incubation conditions, only 13.4 +/- 4.6% (n = 4) of total radioactivity was transferred from [125I]Lp(AI w AII) to Lp(AI w/o AII). The S.A. of A-I after incubation was 2-fold higher in particles with A-II than in particles without A-II. These phenomena were also observed with iodinated high-density lipoproteins (HDL) isolated by ultracentrifugation and subsequently subfractionated by immunoaffinity chromatography. However, when Lp(AI w AII) radiolabeled by in vitro exchange with free [125I]A-I was incubated with unlabeled Lp(AI w/o AII), the S.A. of A-I in particles with and without A-II differed by only 18% after incubation. These data are consistent with the following: (1) in both populations of HDL particles, some radiolabeled proteins transferred rapidly (minutes or less), while others transferred slowly (hours); (2) when Lp(AI w AII) and Lp(AI w/o AII) were directly iodinated, all labeled A-I in particles without A-II were transferable, but some labeled AI in particles with A-II were not; (3) when Lp(AI w AII) were labeled by in vitro exchange with [125I]A-I, considerably more labeled A-I were transferable. These observations suggest the presence of non-transferable A-I in Lp(AI w AII).     

Altered particle size distribution of apolipoprotein A-I-containing lipoproteins in subjects with coronary artery disease

Plasma high density lipoproteins (HDL) can be separated into two subpopulations of apolipoprotein A-I-containing particles: those that also contain apoA-II [Lp(AI w AII)] and those that do not [Lp(AI w/o AII)]. These particles were isolated by immunoaffinity chromatography from 17 men (9 normolipidemic (NL), 8 hyperlipidemic (HL) with symptomatic coronary artery disease (CAD), from 17 NL men without any symptoms of CAD (healthy controls), and from 10 NL men with entirely normal coronary arteriograms (CAD-free controls). The distributions of particle size in these two subpopulations were determined by gradient gel electrophoresis and densitometric scanning. Approximately half of the Lp(AI w AII) particles in all subjects were distributed in the 8.2-9.2 nm interval. For patients with CAD, a greater fraction of the particles were small, in the 7.0-8.2 nm interval [33% in CAD vs. 26% in CAD-free controls (P less than 0.01) and 19% in healthy controls (P less than 0.0001)], and a smaller fraction of the particles were in the 9.2-11.2 nm interval (14% in CAD vs. 24% in CAD-free control (P less than 0.002) and healthy control groups (P less than 0.001). The Lp(AI w/o AII) of both control groups were primarily composed of two discrete subpopulations in the 8.2-9.2 nm and the 9.2-11.2 nm intervals. In CAD patients there were fewer particles in the 9.2-11.2 nm size interval (23% in CAD vs. 33% in CAD-free controls (P less than 0.005) and 36% in healthy controls (P less than 0.0001), and more particles in the smallest 7.0-8.2 nm size interval (32% in CAD vs. 23% in CAD-free controls (P less than 0.01) and 18% in healthy controls (P less than 0.001]. Thus, the spectrum of HDL particle sizes in patients with CAD tends to be shifted toward the smaller particle when compared with the two control groups. This was observed in both NL and HL patients with HDL cholesterol (CH) values in the normal range. As a group, CAD patients had lower HDL (42 +/- 7 mg/dl) and HDL2 (6 +/- 4 mg/dl) CH than healthy (HDL: 49 +/- 7, HDL2: 12 +/- 6 mg/dl) and CAD-free (HDL: 51 +/- 9, HDL2: 12 +/- 6 mg/dl) controls. When controls and patients were compared for their frequencies of abnormal HDL CH levels and particle sizes, abnormalities in HDL and HDL2 CH levels were not significantly more frequent (twofold) among CAD patients than among controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

A mutation in the human apolipoprotein A-I gene. Dominant effect on the level and characteristics of plasma high density lipoproteins

Epidemiologic and genetic data suggest an inverse relationship between plasma high density lipoprotein (HDL) cholesterol and the incidence of premature coronary artery disease. Some of the defects leading to low levels of HDL may be a consequence of mutations in the genes coding for HDL apolipoproteins A-I and A-II or for enzymes that modify these particles. A proband with plasma apoA-I and HDL cholesterol that are below 15% of normal levels and with marked bilateral arcus senilis was shown to be heterozygous for a 45-base pair deletion in exon four of the apoA-I gene. This most likely represents a de novo mutation since neither parent carries the mutant allele. The protein product of this allele is predicted to be missing 15 (Glu146-Arg160) of the 22 amino acids comprising the third amphipathic helical domain. The HDL of the proband and his family were studied. Using anti-A-I and anti-A-II immunosorbents we found three populations of HDL particles in the proband. One contained both apoA-I and A-II, Lp(A-I w A-II); one contained apoA-I but no A-II, Lp(A-I w/o A-II); and the third (an unusual one) contained apoA-II but no A-I. Only Lp(A-I w A-II) and (A-I w/o A-II) were present in the plasma of the proband's parents and brother. Analysis of the HDL particles of the proband by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two protein bands with a molecular mass differing by 6% in the vicinity of 28 kDa whereas the HDL particles of the family members exhibited only a single apoA-I band. The largely dominant effect of this mutant allele (designated apoA-ISeattle) on HDL levels suggests that HDL particles containing any number of mutant apoA-I polypeptides are catabolized rapidly.     

Diaphragm capillarity and oxidative capacity during postnatal development.

In the cat diaphragm, fiber capillarity, cross-sectional area, and succinate dehydrogenase (SDH) activity were measured across the first 6 wk of postnatal development. Fibers were classified as type I, IIa, IIb, or IIc on the basis of staining for myofibrillar adenosinetriphosphatase (ATPase). Capillaries were identified in sections stained for ATPase at pH 4.2. Fiber cross-sectional areas and SDH activities were quantified using an image-processing system. During postnatal development, the proportions of type I fibers increased while type II fibers decreased. At birth, all type II fibers were IIc. From the 1st to the 2nd postnatal wk, the proportion of type IIc fibers decreased while the numbers of IIa and IIb increased. Thereafter the proportion of type IIb fibers continued to increase while the number of IIa steadily declined. At birth, capillarity, cross-sectional areas, and SDH activities of type I and II fibers were low compared with other postnatal age groups. Fiber cross-sectional areas increased progressively with age. The number of capillaries surrounding type I and II fibers increased markedly by the 2nd wk and then continued to increase at a slower rate. The number of capillaries per fiber area reached a peak by the 2nd wk and then declined as fiber cross-sectional area increased. Postnatal changes in capillarity depended on fiber type, being greatest in IIb. SDH activities of type I and II fibers were initially low during the first 2 postnatal wk and then peaked by the 3rd wk. After the 6th wk, fiber SDH activities decreased to adult values. Among the type II fibers, IIb showed the greatest change in SDH activity during early postnatal development.     

Absence of correlation between rates of cell wall turnover and autolysis shown by Bacillus subtilis mutants.

Bacillus subtilis mutants with reduced rates of cell wall autolysis reached a constant rate of wall turnover after a longer lag than the standard strain but eventually showed the same turnover rate. In reverse, a turnover-deficient mutant autolysed at a slightly higher rate than the standard strain. Consequently, there is no correlation between the rates of cell wall turnover and autolysis.     

Distribution of55Fe in juvenile adult lampreys,Petromyzon marinus L., following oral intubation of the isotope

Summary The capacity of a sanguivorous lamprey,Petromyzon marinus L., to deal with ingested iron was studied over time using autoradiography and scintillation counting of solubilized tissue samples after intubation of the oesophagus with a single dose of⁵⁵ferrous citrate. A highly efficient mechanism for absorption in the anterior intestine was recognized with 17% of the intubated radioactivity absorbed into the body after only 5 min, 66% by 3 h, and almost 80% by 21 h. Iron concentration in the epithelial cells of the anterior intestine may be a factor in restricting iron absorption during spontaneous feeding. A decline in total body radioactivity over the 15 days following iron intubation probably results from transport of the metal in the blood and release of radioiron from the mucous cells of the posterior intestine. The kidneys appear to play a smaller but still significant role in iron loss. Gradual increases in radioiron concentration (cpm g^–1 wet weight) and percent of total body radioactivity occur in the liver (2 to 26%), carcass (14 to 37%), and integument (4 to 12%) during the course of the experiment, indicating that these are the chief sites of iron storage during times of metal excess. However, eventually integument may also be a site of iron excretion. Significant fluctuations in radioiron concentration (cpm ml^–1) in whole blood during the 15 day period can be correlated with transport of the metal to sites of storage and excretion, and maybe with incorporation into haemoglobin and with erythropoietic activity. Feeding adult lampreys represent a valuable system, with both general and unique characters, for studying iron metabolism in vertebrates.