Protein Mass Ratio Research Articles

Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism.

Carbon-carbon bond formation is an essential reaction in organic chemistry and the use of aldolase enzymes for the stereochemical control of such reactions is an attractive alternative to conventional chemical methods. Here we describe the crystal structures of a novel class II enzyme, 2-dehydro-3-deoxy-galactarate (DDG) aldolase from Escherichia coli, in the presence and absence of substrate. The crystal structure was determined by locating only four Se sites to obtain phases for 506 protein residues. The protomer displays a modified (alpha/beta)(8) barrel fold, in which the eighth alpha-helix points away from the beta-barrel instead of packing against it. Analysis of the DDG aldolase crystal structures suggests a novel aldolase mechanism in which a phosphate anion accepts the proton from the methyl group of pyruvate.

Serum antibody responses of weanling mice and two-year-old children to pneumococcal-type 6A–protein conjugate vaccines of differing saccharide chain lengths

Type 6A pneumococcal polysaccharide (PS) fractions having mean lengths 2, 7, or 14 repeat units were coupled to tetanus toxoid, giving conjugates T-6A-2, T-6A-7 or T-6A-14. In weanling outbred mice, type-6A serum antibody responses were minimal to T-6A-7 but significantly higher to T-6A-2 and T-6A-14. In contrast, two-year-old humans responded increasingly to T-6A-2, T-6A-7, and T-6A-14; type 6B antibody rose, but variably. Upon challenge with 6B PS at age 3 yr, conjugate-primed children made anamnestic responses. T-6A-14, containing the longest saccharide chains (and highest saccharide:protein mass ratio) excelled in priming as well as antibody induction.

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP.

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.

Characterization of six in vitro reactions mediated by human cytochrome P450: application to the testing of cytochrome P450-directed antibodies.

The identification of cytochrome P450 (CYP) isozymes mediating metabolic pathways of drugs has become increasingly important in anticipating pharmacokinetic drug interactions. The activity of individual CYPs can be monitored in vitro with human liver microsomes by means by relatively specific metabolic reactions: for CYP1A1/2, phenacetin O-deethylation; for CYP2C9, phenytoin 4-hydroxylation; for CYP2C19, S-mephenytoin 4'-hydroxylation; for CYP2D6, dextromethorphan O-demethylation; for CYP3A3/4, alprazolam 4-hydroxylation, and for CYP2E1, chlorzoxazone 6-hydroxylation. We determined the kinetic parameters (Vmax, Km) of these reactions and utilized them to test a monoclonal rat liver CYP1A1 antibody, a monoclonal rat liver CYP2E1 antibody, a polyclonal rabbit anti-rat liver CYP3A1 antibody, and a polyclonal goat anti-rat liver CYP2C11 antibody for their specificity and inhibitory capacity. The CYP1A1 monoclonal antibody (MAb), the CYP2E1 MAb, and the CYP3A1 polyclonal antibody (PAb) inhibited only their respective index reactions. The CYP2C11 PAb inhibited both phenytoin 4-hydroxylation and S-mephenytoin 4'-hydroxylation. At a microsomal versus antibody protein mass ratio of 1:15, 4-hydroxyalprazolam formation was reduced by 73.4% of control with the CYP3A1 PAb; 4'-hydroxymephenytoin formation decreased by 66.3% and 4-hydroxyphenytoin formation by 43.4% with the CYP2C11 PAb; phenacetin O-deethylation was reduced by 39.7% with the CYP1A1 MAb, and 6-hydroxychlorzoxazone formation decreased by 30.0% with the CYP2E1 MAb. Thus, all antibodies tested are at least CYP subfamily specific. The PAbs exhibited greater than 60% inhibition versus the CYP3A3/4- and the CYP2C19-mediated reactions, whereas the MAbs produced less than 50% inhibition for their respective index reactions. Because of their limited inhibitory capacity, MAbs may be correspondingly limited as tools for identification of human CYP enzymes via immunoinhibition studies.

Effect of Fat and Protein Contents on Droplet Size and Surface Protein Coverage in Dairy Emulsions

Oil in water emulsions were prepared using anhydrous milk fat and skim milk homogenized with a laboratory-scale microfluidizer. The fat content was varied between that of raw milk and cream, 4 to 30 g/100g. Protein content was also varied between 3.2 and .4 g/100g; emulsions had a fat to protein mass ratio between 1 and 13. Droplet size distribution and protein surface coverage were measured and expressed in terms of fat to protein mass ratios. Aggregation appeared with increasing fat to protein mass ratio, whereas the average droplet diameter remained unchanged. Surface area in emulsions increased with fat content because of a larger number of droplets rather than a change in droplet size. The fraction of adsorbed protein increased with fat to protein mass ratios; the maximum fraction of adsorbed protein was 85%; therefore, part of the protein present did not adsorb. Protein surface coverage was ca. 10 mg/m2 at fat to protein mass ratios <4 and slightly decreased for higher fat to protein mass ratios.

A New Crystal Form of Proteinase A, a Non-Pepsin-Type Acid Proteinase from Aspergillus niger var. macrosporus1

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase, whose catalytic residues and mechanism remain to be elucidated. A new form of proteinase A crystals more suitable for crystallography than that obtained previously was prepared from an ammonium sulfate solution at pH 3.5 by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1), with unit cell dimensions of a = 69.75 +/- 0.06 A, b = 87.55 +/- 0.05 A, and c = 60.83 +/- 0.04 A. On the assumption of two enzyme molecules per asymmetric unit, the calculated volume to unit protein mass ratio (Vm) was 2.08 A3/Da. By assuming the specific volume to be 0.74 cm3/g, the solvent content (Vso1) was estimated to be 41%, i.e., much larger than that of the crystal form obtained previously at pH 2.0 (Vso1 = 26%). Diffraction data were collected up to a resolution higher than 1.6 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation.

X-ray diffraction analysis of brain lipid membrane structure in Alzheimer's disease and beta-amyloid peptide interactions.

Small angle x-ray diffraction analysis of Alzheimer's disease (AD) lipid membranes reconstituted from cortical gray matter showed significant, reproducible structure changes relative to age-matched control samples. Specifically, there was an average 4 A reduction in the lipid bilayer width and marked changes in membrane electron density profiles of AD cortical samples. There were no significant structure differences in the membrane bilayers isolated from an unaffected region (cerebellum) of the AD brain. Lipid and protein analysis of six AD and six age-matched controls showed that the phospholipid:protein mass ratio was unchanged, but that the unesterified cholesterol:phospholipid (C:PL) mole ratio decreased by 30% in the AD temporal gyrus relative to age-matched controls. The C:PL mole ratio was not significantly different for samples prepared from cerebellum of AD versus control patients. X-ray diffraction analysis of a cholesterol-enriched AD sample demonstrated a virtual restoration of the normal membrane bilayer width and electron density profile, suggesting that the cholesterol deficit played a major role in the AD lipid membrane structure perturbation. Addition of beta-amyloid peptide to bovine brain phospholipid membranes significantly changed the electron density associated with the hydrocarbon core. Alterations in the composition and structure of the membrane bilayer may play an important role in the pathophysiology of AD by altering the activity and catabolism of membrane-bound proteins, including the beta-amyloid precursor protein.

Evidence for changes in the Alzheimer's disease brain cortical membrane structure mediated by cholesterol

—Small angle X-ray diffraction analysis of Alzheimer's disease (AD) lipid membranes extracted from cortical gray matter showed significant, reproducible structure changes relative to age-matched control samples. Specifically, there was an average 4 Å reduction in the lipid bilayer width and significant changes in the membrane electron density profiles of AD cortical samples. There were no significant structure differences in the membrane bilayers isolated from an unaffected region (cerebellum) of the AD brain. Lipid and protein analysis of 6 AD and 6 age-matched controls showed that the phospholipid:protein mass ratio was unchanged but that the unesterified cholesterol:phospholipid (C:PL) mole ratio decreased by 30% in the AD temporal gyrus relative to age-matched controls. By contrast, the C:PL mole ratio in the cerebellum did not change significantly. X-ray diffraction analysis of a cholesterol enriched AD sample demonstrated a virtual restoration of the normal membrane bilayer width and electron density profile, suggesting that the cholesterol deficit played a major role in the AD lipid membrane structure perturbation. Alterations in the composition and structure of the membrane bilayer may play an important role in the pathophysiology of AD by altering the activity and catabolism of membrane-bound proteins, including the β-amyloid precursor protein.

Uptake and metabolism of high-density lipoproteins by cultured rabbit hepatocytes

The selective uptake and internalization of core components of high-density lipoproteins (HDL) were examined in primary monolayer cultures of rabbit hepatocytes. Using [ 14C]sucrose as a surface marker covalently attached to apolipoprotein and [ 3H]cholesteryl linoleyl ether as a core marker, there was a 5–6-fold greater internalization of cholesteryl ether than sucrose-labeled apolipoprotein during 48 h of culture. The rate of uptake of [ 3H]cholesteryl linoleyl ether was 263 ± 29 ng apo HDL/mg cell protein per h during the initial 8 h of culture, but averaged 101 ± 32 ng apo HDL/mg cell protein per h over the 48 h culture period. Concomitant with this apparent selective uptake of cholesteryl ester core, there was a change in the HDL size distribution, with the appearance of a distinct population of smaller 4.3 nm radius particles in addition to the originally predominant particles of 4.9 nm radius. This was associated with a significant reduction of cholesteryl ester as a percentage of lipoprotein mass from 15.5 ± 1.2 to 11.0 ± 1.2 ( P < 0.001) and a reduction in cholesteryl ester: protein mass ratio from 0.30 ± 0.01 to 0.19 ± 0.01 ( P < 0.001). There was no change in the mass ratio of HDL triacylglycerol to protein. Thus rabbit hepatocytes in culture exhibit the capacity to selectively extract cholesteryl ester from HDL and produce smaller HDL particles.

Metabolism of high density lipoproteins by the perfused rabbit liver.

The role of the liver in the catabolism of high density lipoproteins (HDL) was examined in isolated perfused rabbit livers. Using 125I-labeled rabbit HDL the disappearance of labeled apolipoproteins from the perfusate was biphasic with 7% of the label removed after 20 min and a further 6% between 20 and 90 min. In contrast, with HDL labeled with [3H]cholesteryl esters 35% of label had been removed after 90 min. The effect of liver perfusion on HDL size and composition was further studied by recirculating rabbit HDL for 120 min. In control experiments HDL was incubated at 37 degrees C for 120 min with nonperfused media and with media that had been liver perfused. The added HDL was predominantly particles of 4.8-4.9-mm radius, and incubation with nonperfused and preperfused media produced no significant change in size. However, liver perfusion resulted in particles predominantly 4.2-4.3-mm radius. Hepatic perfusion also significantly reduced HDL cholesteryl ester composition as a percentage of lipoproteins mass from 13.3 +/- 2.2% in control incubations to 10.7 +/- 3.1% (p less than 0.001), and cholesteryl ester:protein mass ratio was reduced from 0.31 +/- 0.06 in control to 0.24 +/- 0.10 (p less than 0.001) after 120 min of liver perfusion. Thus interaction of rabbit HDL with rabbit liver results in smaller HDL particles significantly depleted of core cholesteryl esters.

Mechanism of tobacco mosaic virus assembly: Incorporation of 4S and 20S protein at pH 7.0 and 20°C

The mechanism of assembly of tobacco mosaic virus (TMV) has been investigated at pH 7.0 and 20 degrees C by analytical ultracentrifugation. Under these conditions the overall rates of interconversion of 4S and 20S TMV coat protein are sufficiently slow to make possible measurements of the concentrations of remaining 4S and 20S TMV coat protein after addition of homologous RNA to solutions containing, initially, various mass ratios of 20S protein to 4S protein. It has been possible to measure, by schlieren boundary analysis, the relative rates of incorporation of 4S and 20S TMV protein into the growing nucleoprotein rod over the range of initial 20S:4S protein mass ratios from 93:7 to 18:82. The results show that the amount of incorporation of 20S TMV protein depends on the initial 20S:4S mass ratio between approximately 100% and 60% 20S protein but that reconstitution can proceed with approximately 100% 20S TMV protein to form full virus-size rods. However, when the initial protein solutions have less than 60% 20S protein, approximately 80% of the reconstituted nucleoprotein is preferentially formed from 4S coat protein. The remaining approximately 20% appears to require preformed 20S coat protein. These results suggest that a larger region of RNA than previously estimated is involved in the rate-limiting nucleation step in assembly and may explain previously conflicting results concerning the elongation phase of assembly when starting with partially assembled rods.

Stoichiometry and molecular weight of the minimum asymmetric unit of canine renal sodium and potassium ion-activated adenosine triphosphatase.

Sodium and potassium ion-activated adenosine triphosphatase is known to be composed of at least two different polypeptides, alpha and beta. When a detergent-treated supernatant preparation of the enzyme is reacted with the cross-linking reagent, cupric phenanthroline, a single, covalent heterodimer is formed. This product is formed from one of each of the two polypeptides. The remaining, unreacted alpha and beta chains maintain a constant ratio to each other throughout the reaction. The same heterodimer is formed in membrane-bound enzyme when reacted with several other cross-linking reagents. The protein mass ratio between the chains in the native enzyme, determined by two methods, is 2.15 +/- 0.16. Using this value and a value of 121,000 +/- 6,000 for the molecular weight of the larger polypeptide, a molecular weight of 56,000 +/- 7,000 can be calculated for the protein portion of the smaller polypeptide. Upon removal of a substantial portion of the carbohydrate from the smaller polypeptide, a change in its electrophoretic mobility is observed, while that of the larger polypeptide remains unaffected. The apparent length of this unglycosylated small chain is 450 residues, corresponding to a molecular weight of 51,000. Taken together, these results demonstrated that the two polypeptides of the (Na+ + K+)-ATPase exist in an equimolar, noncovalent association in the native enzyme, and that the protein molecular weight of the minimum asymmetric unit is 177,000 +/- 13,000, Previous results which address the question of the quaternary structure of the ATPase are re-examined in light of these determinations.

Association of the major coat protein of fd bacteriophage with phospholipid vesicles

The association of the major coat protein of fd bacteriophage with a phospholipid bilayer was investigated by analyzing the protein's susceptibility to proteolysis and its circular dichroism spectrum when incorporated into single-walled phospholipid vesicles. In the limits tested, this association appeared to be independent of the mass ratio of protein to lipid and of vesicle size, phospholipid composition, and method of preparation. The circular dichroism data are consistent with a similar “membrane-bound” conformation for all cases of vesicle-associated coat protein and for deoxycholate micelle-associated coat protein. Proteolysis of coat protein associated with deoxycholate micelles and with phospholipid vesicles defined the central hydrophobic core presumed to represent that portion of the protein which associates with membrane bilayers in vivo. The isolated core, which assumed a predominantly β-type conformation in detergent solution, maintained a β conformation when associated with a vesicle phospholipid bilayer.

Nuclear acidic protein of the developing Oncopeltus embryos

During the early development of Oncopeltus fasciatus, the nuclear acidic proteins change in rate of synthesis, in amount, and in type. The rate of nuclear acidic proteins synthesis becomes maximal at the 45th h of development. The mass ratio of nuclear acidic protein to DNA increases to its highest level at the 68-h stage and decreases to a constant value after 120 h of development. The amount of nuclear acidic protein per embryo increases linearly from 20 h to 68 h and levels off after 120 h of development. The number of nuclear acidic protein subcomponents increases gradually during the course of development, the emergence of some nuclear acidic protein components being stage-specific. Before or at the beginning of differentiation, the biosynthetic activity is selective. After differentiation the activity is uniformly decreased. The significance of these changes in nuclear acidic proteins and their possible correlations to differentiation are discussed.

Free amino acid pools in the developing sea urchin Lytechinus variegatus

During the early development of Oncopeltus fasciatus, the nuclear acidic proteins change in rate of synthesis, in amount, and in type. The rate of nuclear acidic proteins synthesis becomes maximal at the 45th h of development. The mass ratio of nuclear acidic protein to DNA increases to its highest level at the 68-h stage and decreases to a constant value after 120 h of development. The amount of nuclear acidic protein per embryo increases linearly from 20 h to 68 h and levels off after 120 h of development. The number of nuclear acidic protein subcomponents increases gradually during the course of development, the emergence of some nuclear acidic protein components being stage-specific. Before or at the beginning of differentiation, the biosynthetic activity is selective. After differentiation the activity is uniformly decreased. The significance of these changes in nuclear acidic proteins and their possible correlations to differentiation are discussed.