Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein

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Highlights

• •Zero-length chemical cross-linking of APOA1 peptides in HDL.
• •Cross-links match antiparallel isomers of APOA dimers in molecular modeling.
• •Identical MS/MS spectra of native and synthetic cross-linked peptides.
• •First biochemical evidence of LL5/5 and LL5/4 isomers in human HDL.

     

Mass spectrometric analysis of rat hemoglobin by FAB-overlapping: Primary structure of the α-major and of four β constitutive chains

• 1.1. The globin chain components of Sprague-Dawley rat hemoglobin were obtained by reverse-phase HPLC which showed the presence of two α-chain and four β-chains.
• 2.2. The accurate molecular weight of each globin chain was determined by means of electrospray mass spectrometry. Extensive mass spectrometric analysis on several enzymatic digests by fast atom bombardment mass spectrometry (FAB-overlapping) meant to determine the complete sequence of the α-major and of the four β-globins.
• 3.3. The primary structure of the α-major globin was found in agreement with literature data (Garrick et al., 1975 Biochem. J.149, 245–258; Chua et al., 1987).
• 4.4. Sequence analysis of the four β -globin chains showed that amino acid differences are restricted to two protein portions: the region 22–25 and 123–125, the remaining portions of the molecule being unchanged in the four globins. Furthermore, all the amino acid replacements correspond to single point DNA mutations and (with the exception of the substitution Asp 22 → Asn in the β₂-globin) involve uncharged substitutions.

     

Comparison of the intravascular metabolism of cholesteryl esters and apoproteins of plasma low- and high-density lipoproteins in the rat (Rattus norvegicus), an animal species without plasma cholesteryl ester transfer activity

• 1.1. The intravascular metabolism of the cholesteryl esters (CE) and apoproteins of low density lipoproteins (LDL) and high density lipoproteins (HDL) was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA).
• 2.2. The apoproteins and the CE of LDL had identical catabolic rates, and there was no transfer of LDL CE to other lipoprotein classes.
• 3.3. The CE of the HDL, however, had higher catabolic rates than the apoproteins, and there was transfer of HDL CE to LDL but not to very low density lipoproteins.

     

Effect of bovine milk antigens and egg lysozyme on the binding of 59Fe-lactoferrin to platelet plasma membranes

• 1.1. Platelets bind specifically to lactoferrin. A significant similarity between human lactoferrin and some bovine milk proteins has been established.
• 2.2. Because of the structural homology of lactoferrin and cows milk proteins they are able to influence lactoferrins regulatory function on the level of its binding to membrane receptors on platelets.
• 3.3. An inhibitory effect of bovine α-lactalbumin and of β-lactoglobulin on lactoferrin-receptor interaction was shown.
• 4.4. Bovine α-lactalbumin competes with lactoferrin for the binding sites.
• 5.5. Scatchard plot analysis of data shows one binding site for lactoferrin in the presence of α-lactalbumin with an affinity constant, Ka = 0.46 × 10⁹ mol/1 and 335 receptors/cell.
• 6.6. The inhibitory effect of β-lactoglobulin reaches 62% and is different for the common fraction ⨿-lactoglobulin and the genetic variants β-lactoglobulin A and B.
• 7.7. β-lactoglobulin does not compete with lactoferrin for the membrane receptors.
• 8.8. Bovine casein and egg lysozyme stimulate ⁵⁹Fe-lactoferrin binding to the receptors. The mechanism of these effects is still unknown.
• 9.9. Tested alimentary antigens are able to interact with lactoferrin and also with some platelet membrane structures.
• 10.10. Established changes in lactoferrin binding to the platelet membrane might be in relation to lactoferrins regulatory function and (or) eliminating mechanisms of these alimentary antigens.

     

Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera

• 1.1. A complex of extracellular amylolytic enzymes produced by Saccharomycopsis fibuligera KZ, grown on fine fibre (waste product from corn starch production) and corn-steep liquor, has been studied.
• 2.2. α-Amylases and glucoamylases, as the main representatives of this complex, were separated by hydrophobic chromatography on Spheron 300 LC.
• 3.3. Individual isoenzymes of one type were separated on FPLC-Mono Q.
• 4.4. The relative molecular weight of α-amylases is 54,000, glucoamylases 62,000, maximal activity is reached by both enzymes between pH 5.0 and 6.2 at a temperature of 40–50°C.
• 5.5. Glucoamylases have a higher stability of the native structure than α-amylases, they retain 55% of their original activity, even after 10 min of incubation at 100°C.

     

Main serum protein of rainbow trout (Salmo gairdnerii): its biological properties and significance

• 1.1. Main serum α₁-protein (α₁P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied.
• 2.2. α₁P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results.
• 3.3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition.
• 4.4. Dye- or ConA binding activity.
• 5.5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity.
• 6.6. Possible osmotic regulator.
• 7.7. Significant elevation of blood α₁P level in the course of hepatoma induction.

     

Ontogenic change of digestive enzymes in Penaeus monodon

• 1.1. Ontogenic changes of both proteases and carbohydrases of Penaeus monodon from different larva stages to adult were investigated.
• 2.2. Total protease activity was low during nauplius and zoea but peaked up in mysis. This was due to the activity increase of both trypsin and chymotrypsin.
• 3.3. The change of isozyme pattern of these two enzymes from different life stages of the shrimp was further determined by functional staining on an electrophoregram.
• 4.4. Activity of α-amylase increased after the post-larva stage, while that of chitinase and maltase showed a peak in zoea then gradually decreased to adult.
• 5.5. The ratio of α-amylase activity to protease coincided with the dietary change of the shrimp in different life stage.

     

Thermal stability of fish myosin

• 1.1. Thermal stability of fish myosin has been studied by using differential scanning calorimetry (DSC) and circular dichroism (CD).
• 2.2. The temperature range of the sharp decrease in α-helical content agreed very closely with that of the endothermic peaks.
• 3.3. There was a high correlation between the enthalpy of denaturation (ΔH) and the decreasing quantity in α-helicity (Δh).
• 4.4. The structure of fish myosins was much more unstable than that of rabbit.
• 5.5. The instability of fish myosins was reflected in its rod moiety.

     

Ostrich α-amylase: Purification and characterization of the pancreatic isoenzymes

• 1.1. Four ostrich pancreatic α-amylase isoenzymes were isolated by isoelectric focusing, following affinity chromatography on cyclohepta-amylose-Sepharose 4B.
• 2.2. Amino acid compositions of the four isoenzymes are very similar with only one charged amino acid (Arg) being significantly different.
• 3.3. The molecular weights, as determined by SDS-PAGE and amino acid composition, are nearly identical (52–53 kDa) for all four isoenzymes.
• 4.4. The four α-amylase isoenzymes appear to be kinetically distinct enzymes with a requirement for calcium.
• 5.5. Ostrich α-amylase isoenzymes appear to be non-glycosylated and contain one free thiol group.

     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

A lipid transfer particle in Musca domestica haemolymph

• 1.1. In Musca domestica haemolymph a lipid transfer particle (LTP) is present.
• 2.2. Musca domestica LTP is able to catalyze the transfer of lipids between different housefly lipophorin forms and also between lipophorins of Diptera and Lepidoptera.
• 3.3. The lipophorin of larval Dione juno (Lepidoptera) was purified and is composed of two apolipoproteins, apolipophorin I (M_{r = 209,000}) and apolipophorin II (M_{r = 85,000}) with a density of 1.124 g/ml.
• 4.4. The density of housefly lipophorin undergoes variations during the gonotrophic cycle.
• 5.5. The lipophorin density variation results suggest that when a high rate of lipid utilization occurs, the lipophorin has a higher density value.

     

Renin-angiotensin system in antarctic fishes

• 1.1. The presence of a renin-angiotensin-like system has been investigated in the Antarctic fishes Chionodraco hamatus (Fam. Channichthydae) and Pagothenia (Trematomus) bernacchii (Fam. Notothenidae).
• 2.2. A renin-like activity is present in plasma and kidney of both the white blooded (Chionodraco) and the red blooded (Pagothenia) species.
• 3.3. An angiotensin converting enzyme-like activity has been demonstrated in plasma, gills and kidneys of both species. The activity is inhibited by high temperature.
• 4.4. From our data a renin-angiotensin-like system is present in the Antarctic fishes studied but the cascade of enzymes is active only at low temperatures.

     

Palmitic acid metabolism in hepatopancreas of the freshwater shrimp Macrobrachium borellii

• 1.1. The palmitic acid fate as substrate for the synthesis of either glycerides or other fatty acids was studied in vivo and in the microsomal fraction from hepatopancreas of Macrobrachium borellii.
• 2.2. Most of the palmitic acid administered in vivo circulated to the hepatopancreas, being incorporated mainly in the triacylglycerol (TG) fraction.
• 3.3. Palmitic acid transformations into palmitoleic, stearic and oleic acids were observed in the hepatopancreas.
• 4.4. The in vitro biosynthesis of TG in hepatopancreas was more active than in other tissues. In the microsomal fraction, palmitic acid was also incorporated mainly in TG, and followed the α-glycerophosphate pathway.

     

Neurohypophysial hormones as molecular evolutionary tracers: investigations on the sturgeons Acipenser stellatus and Acipenser guldenstadti

• 1.1. Neurohypophysial hormones of two sturgeon species, Acipenser stellatus and Acipenser guldenstadti, have been purified through molecular sieving on Bio-Gel P4 and reverse-phase high pressure liquid chromatography on Nucleosil C18 columns.
• 2.2. Arginine vasotocin has been identified in both species by its retention time in partition chromatography, amino acid composition and, in the case of A. stellatus, by amino acid sequencing.
• 3.3. A second peptide has been purified and could be α-deamidated vasotocin.
• 4.4. Another peptide with oxytocic activity, distinct from the known oxytocin-like peptides, seems to be present in very small amounts.

     

The effects of artificial solar radiation on wind-stressed tufted titmice (Parus bicolor) and Carolina chickadees (Parus carolinensis) at low temperatures

• 1.1. Changes in metabolic rates and behavior were observed in tufted titmice (Parus bicolor) and Carolina chickadees (Parus carolinensis) exposed to varying conditions of artificial solar radiation, wind, and temperature in a wind tunnel experiment.
• 2.2. During the wind-on condition, both species showed a significant decrease in mean metabolic rates in the high radiation treatments when compared to the low radiation treatments (P < 0.05).
• 3.3. Titmouse orientation, posture and level of activity were significantly affected by radiation and wind conditions.
• 4.4. Metabolic rates observed in the wind tunnel treatments without wind and at low radiation did not significantly differ from similar standard metabolic (black box) treatments (P > 0.05).
• 5.5. Activity levels did not appear to directly affect metabolic rates observed in the wind tunnel treatments.

     

Degradation of bone matrix proteins by osteoclast cathepsins

• 1.1. The degradation of the bone matrix proteins osteocalcin, osteonectin and α₂HS-glycoprotein by human cathepsins B and L and human osteoclastoma cathepsins has been investigated.
• 2.2. Intermediate degradation products (M_r > 12kDa) were not observed during the digestion of α₂HS-glycoprotein and osteonectin by cathepsins B and L although they were observed with some of the osteoclastoma cathepsins. Most of the osteoclastoma cathepsins were capable of degrading these two proteins to small peptides at comparable rates.
• 3.3. Each cathepsin produced a different pattern of osteocalcin degradation products.
• 4.4. The extensive range of non-collagenous proteins in bone matrix may necessitate the production by osteoclasts of cathepsins with different specificities during bone resorption.

     

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

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Evidence for a charge variant of Cd-BP 14, a cadmium-binding protein from Allolobophora caliginosa (Oligochaeta,Annelida)

• 1.1. In Allolobophora caliginosa, a Cd-binding protein distinct in charge from Cd-BP 14, a Cd-binding protein previously isolated from the same oligochaete species [Nejmeddine et al. (1992) Comp. Biochem. Physiol.101C, 601–605], has been purified by a three-step chromatographic procedure including gel permeation and cation-exchange chromatography.
• 2.2. This Cd-binding protein exists in a monomeric form with a molecular weight of 14 kDa and does not contain carbohydrate.
• 3.3. The purified protein significantly absorbed at 280 nm and its amino acid composition revealed the presence of a high level of aromatic amino acids and a lack of cysteine, indicating that the molecule is distinct from metallothioneins.
• 4.4. By contrast, except for its chromatographic behavior on an ion-exchange chromatography column, the metalloprotein was found to be similar to Cd-BP 14. We thus conclude that it represents a charge-variant of Cd-BP 14.

     

Thermal properties for digestive enzymes of a sub-antarctic beetle,hydromedion sparsutum (coleoptera,perimylopidae) compared to those in two thermophilic insects

• 1.1. Proteolytic, lipolytic, amylolytic and cellulolytic activities were studied in adults of the phytophagous beetle, Hydromedion sparsutum, indigenous to the sub-Antarctic island of South Georgia.
• 2.2. Gastric enzyme activities were measured at experimental temperatures of 5–40°C and results were compared with those obtained from two thermophilic insects, Gryllus bimaculatus and Tenebrio molitor.
• 3.3. Protease and lipase activities in Hydromedion were 10–15 times lower than in Gryllus and Tenebrio.
• 4.4. In the temperature range of 5–15°C, α-amylase activity from Hydromedion was only slightly lower than that from Gryllus.
• 5.5. Hydromedion gut homogenates exhibited a distinct cellulolytic activity, even at a low temperature of 5°C.
• 6.6. Cellulolytic activity in the digestive tract of Hydromedion was confirmed by the evolution of ¹⁴CO₂ after consumption of labelled cellulose.
• 7.7. The thermal properties of digestive enzymes agree well with the role of Hydromedion as primary decomposer in its ecosystem.