Anneke Zweers

Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes

Abstract The substitutions Thr → Ala, Gln → Leu and Pro → Thr or Ala in mammalina α-crystallin A chains (19,830 daltons) are found to increase the electrophoretic mobility in sodium dodecyl sulfate gel electrophoresis. Substitutions between residues of like hydrophobicity and small changes in intrinsic charge of the chain did not alter the mobility. Changes in hydrophobicity appear to influence the binding of sodium dodecyl sulfate, and therefore the mobility, whereas proline may affect the conformation of the sodium dodecyl sulfate-protein complex. These effects may depend on the position of the substitution in the chain. Sodium dodecyl sulfate gel electrophoresis is thus able to detect neutral substitutions not usually visible in regular electrophoresis.

THE PLASMA MEMBRANES OF EYE LENS FIBRES. BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION

Abstract Plasma membranes have been isolated from calf eye lens fibre cells. The purified fraction is characterized by the occurrence of a large number of junctional complexes. The cholesterol phospholipid ratio is approximately 0.7, a value in between the values reported for erythrocyte ghosts and liver plasma membranes. Specific membrane protein components have been separated electrophoretically from structural lens proteins.

Selective reassociation of the crystallins

Abstract Under proper conditions a mixture of all the unfolded polypeptide chains which in the native state compose the soluble crystallins reassociate to the “native polymers” α-, β H -, β L - and γ-crystallin. On the other hand, after dissociation and reassociation α- and β-crystallin polypeptides tend to form rod-like hybrids. The presence of both types of subunits in these complexes has been proven by immunoelectrophoresis and polyacrylamide gel electrophoresis in 6 m -urea.

Internally elongated rodent α-crystallin A chain: Resulting from incomplete RNA splicing?

Abstract αA Ins , an elongated α-crystallin A chain previously observed in rat, was present beside the normal αA chain in mouse, gerbil and hamster, which places its origin at least 30 million years ago. Like in rat the sequences of golden hamster αA Ins and αA were found to be identical, apart from the internal insertion of 22 residues in αA Ins . The hamster chains only differed from the rat chains by a single substitution in the inserted sequence of αA Ins . The origin of αA Ins , by insertion of 22 residues in an otherwise unchanged αA chain, and its rigid evolutionary conservation are most easily explained by assuming the incomplete removal of a putative intervening sequence from the precursor mRNA of αA, leaving an intracistronic insert of 66 nucleotides in part of the eventually translated mRNA.

Synthesis of lens protein in vitro. I. Properties of polyribosomes.

Abstract Polyribosomes, characterized by sedimentation behavior, have been prepared from calf-lens epithelium and outer cortex. The polyribosomes are stable during incubation at 37° or freezing at −30°. The effect of ribonuclease and trypsin is reported. The preparations have incorporation ability for amino acids in vitro with either cell sap derived from calf lens or from rat liver and can be stimulated for phenylalanine incorporation by polyuridylic acid to the same extent as bacterial ribosomes.

Improved Separation of Lm (Low Molecular Weight) Crystallins

An improved separation procedure based on centrifugation and gel filtration for low molecular weight (LM) crystallins is described. The isolated fractions have been analyzed by various gel electrophoretic techniques.

A comparison of avian and reptilian δ-crystallin

1. 1. The eye lens protein δ-crystallin is present in different amounts in different birds and reptiles. It is absent in echidna. 2. 2. Chicken, tegu (Tupinambis teguixin) and turtle (Chelonia mydas) δ-crystallins have similar native and subunit mol. wt. Their amino acid compositions show the same characteristics. 3. 3. From peptide maping and amino acid analyses the rate of evolutionary change of δ-crystallin is estimated to be 2–4 times faster than that of α-crystallin.

Improved separation of the alkaline RNAse-inhibitor from eye lens and electrophoretic demonstration of its subunits.

Summary A purification procedure for the natural inhibitor of alkaline RNAse from calf lenses is described. Electro-elution has been introduced as purification step. Complex formation of the inhibitor with RNAse has been demonstrated by polyacrylamide gel electrophoresis. The complex resists dissociation by sodium dodecyl sulfate, whereas in this detergent the inhibitor splits into two subunits of about 30,000 molecular weight.

Proteolytic activity of partly purified ribonuclease inhibitor from rat liver

Abstract Increasing interest has been focused on ribonuclease inhibitor from rat liver as protecting agent in studies on polyribosomes. Most investigators use either the crude 100000 × g supernatant or a preparation partly purified on DEAE-Sephadex. Whereas partially purified material protects the integrity of the polysomes, it contains a component that interferes with peptide formation. The interfering activity is of proteolytic nature and can be removed by gel filtration on Sephadex G-100. Evidence is provided that at least part of the enzymic activity has to be ascribed to a potent leucine aminopeptidase.