Characterization of chicken liver dihydrofolate reductase after purification by affinity chromatography and isoelectric focusing.

Chicken liver dihydrofolate reductase purified to apparent homogeneity by affinity chromatography contains tightly bound dihydrofolate. The most effective method for removal of the bound substrate is by electrofocusing. This procedure also removes previously unsuspected contaminants. In addition, the isoelectric profile revealed as many as four distinct peaks of enzyme activity. The major peak (pI = 8.4) represents 60–75% of the total activity, is devoid of bound substrate, and exhibits an $\text{A}{}_{280}\text{A}{}_{260}$ ratio approaching 1.9 and a specific activity of 14 units/mg. The peak of activity at the isoelectric point of 7.4 contains bound dihydrofolate. The major isoelectric band is shown to be homogeneous by the usual criteria. Notable features of the amino acid composition include a single cysteine, three tryptophans, and an excess of acidic residues. The N-terminal residue is valine. The molecular weight as determined by sedimentation equilibrium is 22,474. The s_20,w⁰ is 2.07. A frictional coefficient of 1.2 indicates that the enzyme approximates a sphere. Circular dichroism measurements suggest a low α-helical content and a high degree of β-structure. The molar extinction coefficient was determined to be 28,970.     

Structure of the bifunctional and Golgi-associated formiminotransferase cyclodeaminase octamer

Mammalian formiminotransferase cyclodeaminase (FTCD), a 0.5 million Dalton homo-octameric enzyme, plays important roles in coupling histidine catabolism with folate metabolism and integrating the Golgi complex with the vimentin intermediate filament cytoskeleton. It is also linked to two human diseases, autoimmune hepatitis and glutamate formiminotransferase deficiency. Determination of the FTCD structure by X-ray crystallography and electron cryomicroscopy revealed that the eight subunits, each composed of distinct FT and CD domains, are arranged like a square doughnut. A key finding indicates that coupling of three subunits governs the octamer-dependent sequential enzyme activities, including channeling of intermediate and conformational change. The structure further shed light on the molecular nature of two strong antigenic determinants of FTCD recognized by autoantibodies from patients with autoimmune hepatitis and on the binding of thin vimentin filaments to the FTCD octamer.     

On the detection of functional and structural enzyme mutants by coordinated affinity chromatography and isoelectric focusing

A method of studying structural and functional heterogeneity of enzymes has been developed and tested on chymotrypsin. The enzyme, prepared from single mouse pancreata, has been fractionated with respect to function and charge content by a combination of affinity chromatography and isoelectric focusing. By comparing chymotrypsin isolated from isogenic strains, chymotrypsinogen of strains A/Sn and NZB was found to be genetically heterogeneous, thus not revealed as different chymotrypsin forms of a single zymogen. Chymotrypsinogen originating from two loci was investigated, and structural and functional differences of the corresponding enzymes were determined. At both loci, structural allelomorphism was indicated. At one locus, the structural heterogeneity was also found to be reflected in functional heterogeneity of the corresponding enzymes. By mating the two strains and fractionating the enzyme of the cross, the differences were shown to be inherited.     

The purification of Aeromonas aminopeptidase by affinity chromatography

A competitive inhibitor for Aeromonas aminopeptidase has been prepared from the bromomethyl ketone derived from t-butyloxycarbonyl-l-leucine and successfully coupled to aminomethyl cellulose to form an adsorbent for affinity chromatography. The blocked form of the inhibitor was coupled to aminomethyl cellulose and then deblocked in aqueous trifluoroacetic acid to yield an insolubilized analog of an NH₂-terminal l-leucyl residue. This material was effective in binding the aminopeptidase and separating it from a contaminating endopeptidase, which has a similar isoelectric point and size. Separation of the aminopeptidase and endopeptidase was shown to be due to the specificity of the affinity adsorbent for the aminopeptidase, inasmuch as separation of the enzymes did not occur on a typical anion exchange column or on a hydrophobic column lacking a free amino group.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

The purification of the hepatic glutathione S-transferases of rainbow trout by glutathione affinity chromatography alters their isoelectric behaviour.

1. The basic glutathione S-transferases from rainbow-trout liver were more stable than the acidic ones. 2. The apparent pI values of these enzymes were lowered when they were eluted from a glutathione affinity column by reduced glutathione at pH 8.85. 3. The pI effect was not a function of the high pH alone, was diminished under conditions less favourable to glutathione oxidation, and did not occur when S-hexylglutathione affinity chromatography was used instead.     

Fractionation of horseradish peroxidase by preparative isoelectric focusing, gel chromatography and ion-exchange chromatography.

Horseradish peroxidase has been fractionated by preparative isoelectric focusing in a density gradient and in a layer of granulated gel using pH-3-10 and narrow-pH-range carrier ampholytes at different total enzyme loads. The resolution of peroxidase isoenzymes in preparative-layer isoelectric focusing was comparable to that obtained by analytical thin-layer isoelectric focusing. Isoelectrically homogeneous isoenzymes could be isolated with good recovery in a single fractionation step. Despite the excellent separation of the individual isoenzymes by isoelectric focusing in gel layers, an effective purification, indicated by the absorbance ratio A403mn/A278nm, could not be achieved by focusing applied as a single step. By different fractionation sequences combining gel chromatography, ion-exchange chromatography, and isoelectric focusing, individual isoenzymes with a high purity and homogeneous with respect to their size and charge properties have been isolated.     

Isolation and purification of morphine receptor by affinity chromatography

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.     

Partial purification of beta-N-acetylhexosaminidase A by affinity chromatography

A glycopeptide derived from bovine nasal septum by sequential treatment with trypsin, chymotrypsin, 0.05N HCl in dry methanol (desulfation), testicular hyaluronidase and β-glucuronidase, was coupled to Sepharose-4B in the presence of cyanogen bromide. β-$\text{N}$-Acetylhexosaminidase A was selectively retarded when crude extracts of human skin fibroblasts or liver were applied to the affinity column and was subsequently eluted with 0.1% Triton X-100 in 0.1M citrate-phosphate buffer pH 4.4, providing a simple method for purification.     

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).