Loss Of Functional Activity Research Articles

Polyribosome binding of rabbit globin messenger RNA and messenger ribonucleoprotein labelled with bacteriophage-T4 RNA ligase and 5′-[32P] phosphocytidine 3′-phosphate

Rabbit polyribosomal globin messenger RNA (mRNA) and messenger ribonucleoprotein (mRNP) were labelled at the 3' poly(A) tail to high specific activity with T4 RNA ligase and [5'-(32)P]pCp without consequent loss of functional activity. Labelled message was translated in both micrococcal nuclease treated and untreated rabbit reticulocyte lysates, as shown by the formation of labelled polyribosomes. The utilisation of labelled messenger was abolished by T2 toxin or sodium fluoride which are known to inhibit protein synthesis.Images

Iodination of biological samples without loss of functional activity

A technique for radioactive labeling by iodination of sensitive biological material that preserves the functional activity of the samples to a greater extent than the standard iodination methods is presented. The reaction is carried out keeping in separate phases the substrate and the iodine-generating system. Chloramine-T in the presence of water and Cl − ions generates Cl 2 on a piece of filter paper kept close to the surface of the solution containing the substrate and the 125I −. The Cl 2 molecules diffuse from the paper, enter the solution, and, reacting with the iodide ions, generate iodine that modifies the aromatic rings of the sample. Protein A, lysozyme, and ribosomes treated under these iodination conditions are much less affected in their activity than when iodinated by the standard chloramine-T method or the iodogen system. In addition, this technique, which we have called “two phases” system, seems to act preferentially on the surface of the structures as shown by studying the iodination pattern of the ribosomal proteins.

Proteolytic cleavage and inactivation of alpha 2-plasmin inhibitor and C1 inactivator by human polymorphonuclear leukocyte elastase.

This study has examined the interaction between human leukocyte elastase and alpha 2-plasmin inhibitor, or C1 inactivator, inhibitors of proteases of the complement, kinin, coagulation, and fibrinolytic enzyme systems. Leukocyte elastase, in catalytic concentrations, progressively inactivates the plasmin inhibitory activity of both inhibitors. The C1s binding function of C1 inactivator is also destroyed by leukocyte elastase. The nature of the molecular events underlying the inactivation of these protease inhibitors was examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Loss of functional activity was accompanied by limited proteolytic cleavage of both inhibitors with the production of several characteristic derivative peptide chains. Leukocyte elastase cleaved alpha 2-plasmin inhibitor at two separate sites and generated lower molecular weight fragments similar to those produced by bovine beta-trypsin. C1 inactivator was hydrolyzed at three different regions on the molecule whereas beta-trypsin cleaved two regions in common with leukocyte elastase. These findings suggest that inactivation of alpha 2-plasmin inhibitor and C1 inactivator by leukocyte elastase released in the inflammatory reaction may potentiate pathological proteolysis. The limited digestion of these inhibitory proteins by leukocyte elastase may prove useful in studies of their primary structure.

Purification and radiolabeling of human C1q.

A new procedure for isolating human C1q from serum or plasma is described. The method, that is highly selective, rapid and involves minimal handling, yields fully active, immunoglobulin-free unaggregated C1q. Several different methods of radiolabeling C1q are compared. These include two methods selective for tyrosine residues, two that label lysine residues, and a method that labels sialic acid residues. The effect of each of the labeling procedures on C1q hemolytic activity was assessed. Also appraised for each method was the ability of the labeled molecules to bind to antibody sensitized cells and to interact with C1r and C1s to form C1. The distribution of each of the radiolabels among the three polypeptide chains of C1q and between the collagenous and globular regions of C1q was determined. Methods were identified that selectively labeled the globular portion of either the A or C polypeptide chain of the C1q molecule without loss of functional activity. Another of the methods labeled all three polypeptide chains relatively uniformly without significant loss of activity.

Tritium radiolabeling of antibodies to high specific activity with N-succinimidyl[2,3- 3H]propionate: Use in detecting monoclonal antibodies

A simple method has been established for tritium labeling immunoglobulins. This label avoids certain disadvantages of 125iodine such as high radiation hazard and requirement for frequent labeling owing to the short half-life of this isotope. High specific activities (range 1.8–9.0 cpm/μg protein) were obtained with no loss of functional activity as demonstrated by passive hemagglutination. The labeled conjugate is especially suitable for detection of activity in hybridoma supernatants by indirect binding assay. Adapted to microtiter plates, the method is suitable for large numbers of samples, e.g. up to 800 supernatants may be examined daily. The utility and advantages of the binding assay with respect to reproductibility, sensitivity, binding kinetics, easy and rapid performance are described.

Chemical stability of bacteriophage T7 early mRNA.

T7 early mRNA produced by a gene 1 amber mutant phage, T7 am27, is chemically stable interms of acid insolubility and T7 DNA hybridizability. However, the messenger activity of individual T7 early mRNA species, transcripts of gene 1, gene 0.7, and gene 1.3, decay with a half-life of about 6.5 min at 30 C. An extensive secondary structure is present in all T7 early mRNA species and is probably responsible for the chemical stability of the RNAs after the loss of functional activity. It is unlikely that ribosomes protect T7 early mRNA from nucleolytic degradation.

Protein polymorphism in man.

Perhaps the most important contribution that molecular biology has made to population genetics has been in the demonstration of the extraordinary number of alleles wish different properties which may in principle be generated from a single gene by separate mutational events. For example, from a gene containing a sequence of DNA, say 900 bases long and coding for a polypeptide of 300 amiooacids, 2,700 different alleles each differing from the original by only a single base change may be formed by separate mutations, since each of the 9(EO bases may be altered to one of three others in different mutational events. The functional effects of these different alleles can vary very widely. From what is known about rhe genetic code and about the aminoacid composition of proteins we can estimate that some 20-25% of all mutations of this b e will b~synonymous. That is 1:o say they will result in no alteration in the aminoacid structure of the nrotei~n because thev will sirn~lv involve the L J change of one particular codan ;o another specifying the same aminoacid. In about 3-6% of cases, because the mutation involves the alteration of a base triplet coding for an aminoacid to a nonsense triplet which results in chain termination, the mutant allele will cause the synthesis of a shortened polypeptide chain lacking a greater or lesser portion of its carboxyl terminal amin* acid sequence. In most instances this is likely to result in considerable disruption of protein structure with loss of functional activity. However, in some 70-75% of cases, a single base change win result in the synthesis of a protein differing from the original by the substitution of one aminoacid for another at the corresponding point in its aminoacid sequence. Since each of the several hundred aminoacids in a protein may, be changed to one of several others by such mutations, a considerable variety, of structurally different variants may be produced in thi!; way. Furthermore! ,they may be expected to differ in their properties one firom another, accordmg to the particular aminoacid which has been substituted and the particular site in the protein where the substitution has occurred. In the case of enzyme proteins for example, such mutations may in some cases lead to a gross reduction or even a complete loss of activity, while in other cases only minor changes in function or perhaps none at all will be produced. Finally, besides the gene mutations which involve only a change in a single base, an indeterminate number of other kinds of mutational event can also occur. They may result in deletions, duplications or other alterations of the base sequence, and they can be expected to produce corresponding altera-