Alternations in lipid membrane fluidity and the physical state of cell-surface sialic acid in zinc-deficient rat erythrocyte ghosts

Erythrocyte ghosts, prepared from the blood of rats fed zinc-deficient diets, were evaluated for membrane fluidity and surface sialic acid properties using spin-labeled probes and electron spin resonance (ESR) spectroscopy. These physical parameters of the erythrocyte ghosts from the zinc-deficient group were compared to those for erythrocyte ghosts obtained from ad libitum and pair fed controls consuming zinc-adequate diets. As the animals became progressively zinc deficient, the erythrocyte ghost membranes became more fluid than those from the control groups. In addition, the apparent rotational correlation time of Tempamine spin probes on surface sialic acid residues was smaller for the zinc deficient group, indicative of an increased rotational mobility of the spin label. These results suggest that zinc deficiency can have pronounced effects on the physical state of membrane bilayer lipids and cell surface carbohydrates and supports the view that many of the pathological signs of zinc deficiency are due to a general membrane defect.     

The response of murine intestinal crypts to short-range promethium-147 beta irradiation: deductions concerning clonogenic cell numbers and positions

An exteriorized loop of mouse intestine was exposed to 147Pm low-energy electrons, where the dose rate decreased by a factor of 5 from the base of the crypt to the top of the proliferative zone. A crypt survival curve was obtained, expressed in terms of exposure time. The shape of the curve was interpreted in terms of survival parameters for colony-forming cells (clonogens) derived using 137Cs gamma rays and the depth-dose curve measured for 147Pm electrons. It is concluded that the shape of the crypt survival curve using 147Pm electrons is inconsistent with the notion of either the presence of a large number of clonogens or a small number near the top of the proliferative zone. A computer fitting procedure showed that the best agreement between predicted and observed curves was achieved with 2.7 +/- 0.5 clonogens at cell position 5.6 +/- 0.6, in the putative stem-cell zone.     

A redetermination of the concentration of alpha 2-macroglobulin in human plasma

The concentration of alpha 2-macroglobulin in human plasma has been remeasured utilizing a carefully isolated and characterized sample of alpha 2-macroglobulin as a standard. A highly purified sample of alpha 2-macroglobulin with a total trypsin binding capacity of 1.7 mol trypsin/mol alpha 2-macroglobulin was used as a standard for both a radial immunodiffusion and a rocket immunoelectrophoresis technique. With this preparation as a standard, the concentration of alpha 2-macroglobulin in a normal plasma pool over 10,000 donors was found to be about 1.2 mg/ml. A similar concentration (1.3 mg/ml) was found when using a functional trypsin binding assay. This concentration is considerably less than the usually accepted mean of the normal range for alpha 2-macroglobulin.     

Detection of prophospholipase A2 in human spermatozoa

Prophospholipase A2 (proPA2) has been isolated from human spermatozoa after acid extraction and chromatography on hydrophobic WP-Butyl (C4) and ion-exchange (SP 5PW) columns. The addition of benzamidine, a noncompetitive synthetic trypsin inhibitor, to semen samples has kept a portion of the sperm phospholipase A2 (PA2) in its zymogen form and allowed its isolation after acid extraction. When radioactive phosphatidylcholine (PC) or phosphatidylethanolamine (PE) were used as substrates, an identical elution profile of this enzyme was obtained on a C4 column. The proenzyme was separated from active PA2 on the C4 column. Human sperm proPA2 exhibited a less cationic charge than active PA2 on the SP 5PW column. Porcine pancreatic proPA2 had the same chromatographic behavior on high performance liquid chromatography (HPLC) (SP 5PW) as human sperm proPA2. The purification procedure resulted in the isolation of proPA2 which, upon activation by proteolysis, presented the same chromatographic elution profile on HPLC as active PA2 of human spermatozoa and porcine pancreas. Thus, a zymogen form of PA2 exists in human spermatozoa.     

Properties of the interaction between phosphofructokinase and actin

The interaction of rabbit skeletal muscle phosphofructokinase (PFK) with actin is characterized in terms of the binding of PFK to actin in the presence and absence of tropomyosin and troponin, the effect of PFK on actin polymerization, and the involvement of adenylates in the binding of PFK to actin. The thin filament proteins, tropomyosin and troponin, are associated with skeletal muscle actin and reduce the binding of PFK to actin, thus influencing the probable distribution of PFK in skeletal muscle. The binding of PFK to actin is inhibited by ATP and ADP but not by fructose 6-phosphate or fructose 2,6-bisphosphate. This specific inhibition, plus evidence from fluorescence quenching and photoaffinity labeling, suggests that actin binds at the adenosine activation sites of PFK. Light scattering measurements used to monitor actin polymerization indicate that PFK dramatically increases the level of light scattering produced by the polymerization of actin, indicative of a superaggregate of PFK and actin. PFK inhibits the polymerization of actin when polymerization is induced by low concentrations of added salts. Although PFK binds to actin with high affinity, it seems to have little effect on the high shear viscosity of actin filaments.     

Secondary structures of rat lipolytic enzymes: circular dichroism studies and relation to hydrophobic moments

To explore the secondary structures of lingual and pancreatic lipases, circular dichroism measurements were performed. Maximum average ellipticities were used to calculate the percentage of alpha-helices, beta-sheets, and random coils. Lingual lipase had an ellipticity of -20235 +/- 140 deg cm2/dmol (mean +/- SE) at 220 nm suggesting 60% alpha-helix, 20% beta-sheet and 20% random coil structure, but the mean ellipticity for pancreatic lipase was -14093 +/- 82 deg cm2/dmol (mean +/- SE) at 210 nm suggesting a 34.8% alpha-helical, 25% beta-sheet and 40% random coil secondary structure. An alpha-helical stretch of residues with a large hydrophobic moment ("globular" alpha-helix by hydrophobic moment plot) from amino acids 382 through 389 at the COOH-terminal end of lingual lipase was noted. This sequence, absent in pancreatic lipase, may account for the avid binding of lingual lipase to fat emulsion particles.     

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide     

Molecular cloning of the uteroferrin-associated protein, a major progesterone-induced serpin secreted by the porcine uterus, and the expression of its mRNA during pregnancy

The uteroferrin(Uf)-associated basic proteins (UfAP) are a group of three (Mr = 42K, 48K, and 50K) antigenically related, basic glycoproteins secreted by the porcine uterus under the influence of progesterone (P4) which exist as heterodimers (Mr = 80,000) with the iron-binding acid phosphatase, Uf. Several UfAP cDNA clones from a day-60 pregnant pig uterine endometrial cDNA library have been cloned and sequenced. The UfAP mRNA is approximately 1400 bases long and has a single open reading frame of 1251 bases with two start codons at positions 64 and 79 from the 5'-end. UfAP mRNA content of the endometrium increases as pregnancy proceeds, reaching maximum levels around day 70 and then remaining relatively constant in late gestation (days 70 to 110). The pro-form of the UfAP minus signal sequence appears to be 392 amino acids in length and has four potential N-linked glycosylation sites Asn107, Asn197, Asn243, and Asn315. Comparison of the NH2-terminal sequences of the individual UfAP poly-peptides with the amino acid sequence deduced from the cDNA has indicated a series of at least four posttranslational proteolytic processing steps which generate the various molecular forms of the UfAP. The deduced amino acid sequence of UfAP shares considerable identity with several protease inhibitors and hormone-binding proteins that are members of the serpin superfamily of proteins. The UfAP amino acid sequence also exhibits about 55% sequence identity with the P4-induced uterine milk proteins (UTMP) of the sheep. Since the UfAP and UTMP share many biosynthetic and structural features that include site of biosynthesis in the endometrium, P4-responsiveness, the presence of the mannose 6-phosphate lysosomal recognition marker, and considerable sequence similarity, the UfAP and the UTMP may have homologous function which for both still remains obscure.