Biomolecular interaction analysis : strategies for antibody and antigen characterization.

One of the most important classes of reagents for clinical diagnosis is antibodies, either in polyclonal preparations, as monoclonal antibodies (MAbs), or as customized reagents prepared by genetic engineering. The enormous range of antibodies produced by hybridoma and recombinant technologies, together with the requirements of clinical diagnosis for sensitivity and selectivity, make heavy demands on the processes of selecting and characterizing suitable antibody reagents.

Introducing a biosensor based technology for real-time biospecific interaction analysis.

This report describes a system for real-time biospecific interaction analysis, using biosensor technology based on the optical phenomenon surface plasmon resonance. The biospecific interface is a sensor chip consisting of a thin gold film deposited on a glass support and covered with a hydrogel matrix. One component of the interaction being studied is attached covalently to the hydrogel, and other interactants are passed over the chip in solution. The interaction is followed in real time in terms of changes in the mass concentration of biomolecules at the sensor surface. Surface concentrations down to 10 pg/mm2 can be measured. The technique does not require molecular labels such as isotopes or spectroscopic markers, and purification of interacting components can often be avoided. Repeated analyses can be performed on the same sensor chip. With this system, the same general procedure can be used for a wide range of different applications, including concentration determination, kinetic measurements and multi-site binding studies. The sensitivity of the technique can be adjusted by choice of reagents and experimental procedure: determination of specific proteins in serum down to 20 ng/ml and macromolecular association constants from 10(7) M-1 up to 4 x 10(11) M-1 are documentated examples. No other single analytical system has the same versatility and general applicability to biospecific interaction analysis. The system is developed and marketed by Pharmacia Biosensor AB, Sweden.

The primary structure of human platelet profilin: reinvestigation of the calf spleen profilin sequence.

The primary structure of human platelet profilin was determined by aligning the sequences of its tryptic peptides to the previously determined calf spleen profilin sequence [(1979) FEBS Lett. 101, 161-165]. Comparison of the peptide fingerprints of the two proteins suggested a higher homology than that found by direct sequence comparison. We therefore reinvestigated the sequences of the peptides from calf spleen profilin. We identified four incorrect charge assignments and a deletion of three residues. The similarity between the two vertebrate profilins amounts to 95%.

Rapid purification of deoxyribonuclease I using fast protein liquid chromatography.

Deoxyribonuclease I finds extensive application in the fields of both nucleic acid and cell motility research. This paper describes the use of the cationic exchange column MonoQ, marketed by Pharmacia as part of their Fast Protein Liquid Chromatography system, for further purification of the enzyme from commercially available material. Up to 7 mg DNase I of high purity can be obtained in a single separation step taking about 20 min to perform. The quality of the product is documented using 3 independent assay criteria.

A 90 000-dalton actin-binding protein from platelets. Comparison with villin and plasma brevin.

Affinity chromatography of Ca2+-containing extracts of platelets on DNAase I-Sepharose, using Ca2+-free buffer as eluant, selects a 1:1 complex of a 90 000-dalton protein with actin. The complex shows little interaction with either DNAase or actin unless Ca2+ is present. In the presence of Ca2+, the complex nucleates polymerization of actin, reduces the viscosity attained, and delays filament formation from profilactin with characteristics closely resembling those shown by chicken villin. Proteolysis of the native proteins indicates structural similarity between the platelet protein and villin or villin core; limited proteolytic digestion in the presence of SDS distinguishes the platelet protein from villin but not from the functionally related plasma protein, brevin. The platelet protein is not accessible to enzyme-mediated iodination of surface components on intact cells. The term 'platelet brevin' is proposed for the protein.

Nucleation of actin polymerization from profilactin. Opposite effects of different nuclei.

The lag in polymerization of calf spleen profilactin in response to addition of MgCl2 can be overcome by small amounts of spectrin-actin-band 4.1 complex, covalently crosslinked actin oligomers or sonicated F-actin. All of these factors also nucleate polymerization of pure actin. Another nucleator of actin polymerization, villin, delays filaments formation from profilactin. A simple model of the interaction of profilin with actin can explain these apparently conflicting results in terms of the polarity with which actin filaments elongate from the different nuclei.

Characterization of platelet extracts before and after stimulation with respect to the possible role of profilactin as microfilament precursor.

The amount of profilactin in platelet extracts made in the absence of free Ca++ ions decreases and the amount of free profilin increases as a consequence of thrombin stimulation. This agrees with the proposed role of profilactin as a microfilament precursor in nonmuscle cells. Filamentous actin in extracts of unstimulated platelets appears partly in large aggregates that contain actin binding protein (ABP) and relatively few other proteins. After stimulation, the amounts of actin and ABP in the aggregates are increased and myosin is also included together with a few additional proteins. When the cells are lysed in the presence of Ca++, aggregation is drastically reduced. The data indicate that filamentous actin depolymerizes rapidly and recombines with available profilin, and that a Ca-specific interaction also occurs between actin and a new protein with molecular weight about 90,000.

Reorganization of actin in platelets stimulated by thrombin as measured by the DNase I inhibition assay.

The effect of thrombin stimulation on actin organization in human platelets has been analyzed by using the DNase I inhibition assay, which is selective for unpolymerized and filamentous actin. The results provide biochemical evidence for the suggestion that stimulation leads to rapid polymerization of actin. The measurements also reveal changes in the polymerization state of actin occurring after cell lysis. These changes are influenced by the concentration of free calcium in the extracts.

The unpolymerised form of actin in non-muscle cells.

Several lines of evidence point to the existence of unpolymerised actin in non-muscle cells. Ultrastructural examination reveals both a variety of actin filament bundles and actin in a controversial organisational state. Arguments are cited that this material, which at least in part is found close to the plasma membrane, represents unpolymerised actin rather than a random array of single actin filaments. The rearrangement of actin filament bundles during the cell cycle, and in response to experimental manipulation, suggests a turnover of filaments by a polymerisation-depolymerisation cycle. Extracts made from non-muscle cells under conditions where muscle actin would polymerise still contain appreciable fractions of monomeric actin. Studies on purified polymerisation-resistant actin from a variety of sources reveal the presence of a small protein which binds specifically to actin and prevents polymerisation. In the last section of the article, we expand the idea that this auxiliary protein is a central control element in the regulated exchange between non-polymerised and polymerised actin in vivo.

Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I.

A simple and selective assay for monomeric and filamentous actin is presented, based on the inhibition of DNAase I by actin. In mixtures of monomeric and filamentous actin, only the monomeric form is measured as DNAase inhibitor. The total amount of actin in a sample can be determined after depolymerization of F actin with guanidine hydrochloride. The assay is rapid enough to detect changes in the polymerization state of actin in vitro over time intervals as short as 3 min. Data characterizing unpolymerized and filamentous actin pools in extracts of human platelets, lymphocytes and HeLa cells are presented.

The composition of an unusual precursor of 50 S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes. The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.

An unusual precursor of 50S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein ("47S") particles during exponential growth. These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12. Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes.

A 30 S precursor of 30 S ribosomes in a mutant of Escherichia coli.

Escherichia coli 15-28, a mutant with a defect in ribosome metabolism, accumulates a ribonucleoprotein particle that is indistinguishable from 30S subunits by sedimentation but contains the precursor form of 16S RNA. This particle is probably a precursor of 30 S ribosomes.