Graham S. Bailey

Abnormal regulation of renal kallikrein in experimental diabetes. Effects of insulin on prokallikrein synthesis and activation.

The effects of streptozotocin (STZ) diabetes and insulin on regulation of renal kallikrein were studied in the rat. 1 and 2 wk after STZ injection, diabetic rats had reduced renal levels and urinary excretion of active kallikrein. Tissue and urinary prokallikrein levels were unchanged, but the rate of renal prokallikrein synthesis relative to total protein synthesis was reduced 30-45% in diabetic rats. Treatment of diabetic rats with insulin prevented or reversed the fall in tissue level and excretion rate of active kallikrein and normalized prokallikrein synthesis rate. To further examine insulins effects, nondiabetic rats were treated with escalating insulin doses to produce hyperinsulinemia. In these rats, renal active kallikrein increased. Although renal prokallikrein was not increased significantly by hyperinsulinemia, its synthesis was increased. As this was accompanied by proportionally increased total protein synthesis, relative kallikrein synthesis rate was not changed. Excretion of active kallikrein was unchanged, but prokallikrein excretion was markedly reduced. Therefore, increased tissue active kallikrein seen with hyperinsulinemia can be explained not only by increased synthesis but also by retention and increased activation of renal prokallikrein. These studies show that STZ diabetes produces an impairment in renal kallikrein synthesis and suggest that this disease state also impairs renal prokallikrein activation. The findings also suggest that insulin modulates renal kallikrein production, activation, and excretion.

The Production of Antisera

Suitable antisera are essential for use in all immunochemical procedures. Three important properties of an antiserum are avidity, specificity, and titer. The avidity of an antiserum is a measure of the strength of the interactions of its antibodies with an antigen. The specificity of an antiserum is a measure of the ability of its antibodies to distinguish the immunogen from related antigens. The titer of an antiserum is the final (optimal) dilution at which it is employed in the procedure; it depends on the concentrations of the antibodies present and on their affinities for the antigen. The values of those parameters required for a particular antiserum very much depend on the usage to which the antiserum will be put. For example, for use in radioimmunoassay, it is best to have a monospecific antiserum of high avidity, whereas for use in immunoaffinity chromatography the monospecific antiserum should not possess too high an avidity otherwise it may prove impossible to elute the desired antigen without extensive denaturation.

Rapid purification of a prekallikrein from rat pancreas

Abstract A prekallikrein from rat pancreas was purified 1500-fold with an overall yield of 20% using a rapid, simple procedure. DEAE-Sephadex A-50 chromatography permitted the separation of two prekallikrein components present in rat pancreatic homogenates; the major fraction was further purified by Sephadex G-100 gel filtration and immunoadsorption chromatography. The zymogen is a single-chain molecule with pI 4·35. Apparent Mr values of 38,000 and 37,000 were determined by sodium dodecyl sulfate-polyacrylamide gradient electrophoresis and gel filtration, respectively.

Immunodiffusion in Gels

Immunodiffusion in gels encompasses a variety of techniques that are useful for the analysis of antigens and antibodies (1). The fundamental immunochemical principles behind their use are exactly the same as those that apply to antigen#x2013;antibody interactions in the liquid state. Thus an antigen will rapidly react its specific antibody to form a complex, the composition of which will depend on the nature, concentrations, and proportions of the initial reactants. As increasing amounts of a multivalent antigen are allowed to react with a fixed amount of antibody, precipitation occurs, in part because of extensive crosslinking between the reactant molecules. Initially the antibody is in excess and all of the added antigen is present in the form of an insoluble antigen#x2013;antibody aggregate. Addition of more antigen leads to the formation of more immune precipitate. However, a point is reached beyond which further addition of antigen produces an excess of antigen and leads to a reduction in the amount of the precipitate (see Fig. 1) because of the formation of soluble antigen#x2013;antibody complexes. Fig. 1. Variation in the amount of immune precipitate on addition of increasing amounts of antigen to a fixed amount of antibody.

The purification of a bovine kidney enzyme which cleaves melanocyte-stimulating hormone-release inhibiting factor

An enzyme which catalyzes the hydrolysis of L-prolyl-L-leucylglycinamide, the factor which inhibits the release of melanocyte-stimulating hormone, was purified 189-fold from bovine kidney in a 5% yield. The molecular weight of the enzyme on gel filtration was estimated to be 300 000 and its isoelectric point was found to be pH 4.1. The single component seen on sodium dodecyl sulphate-gel electrophoresis was estimated to have a molecular weight of 56 000, indicating that the native enzyme may be a pentamer or hexamer. The enzyme could clearly be distinguished from other prolyl-cleaving enzymes.

Purification and partial characterisation of a bovine kidney aminotripeptidase (capable of cleaving prolyl-glycylglycine).

SummaryAn enzyme was purified 163-fold in an 8.2% yield from bovine kidney. The specific activities of the pure preparation against L-prolyl glycylglycine and L-alanyl glycylglycine were found to be 244.5 and 578 μ, Moles substrate hydrolyzed per min per mg protein respectively. The molecular weight of the enzyme was estimated on gel filtration to be 55,000. The isoelectric point was recorded to be pH 5.2. A preliminary study of substrate specificity showed that the enzyme preferentially hydrolyzed tripeptides of the type X-glycylglycine. The enzyme was tentatively identified as a tripeptide aminopeptidase (α aminoacyldipeptide hydrolase, EC 3.4.11.4).

Purification and specificity of prolyl dipeptidase from bovine kidney

Prolyl dipeptidase (iminodipeptidase, L-prolyl-amino acid hydrolase, EC 3.4.13.8) was purified 180-fold from bovine kidney. The enzyme which was obtained in a 10% yield was completely separated from a number of known kidney peptidases including an enzyme of very similar substrate specificity, proline aminopeptidase (L-prolyl-peptide hydrolase, EC 3.4.11.5). The specific activity of the enzyme with L-prolylglycine as substrate is 1600 units of activity per mg protein. Optimum activity of the enzyme is at pH 8.75 and the molecular weight on gel filtration was estimated to be 100 000. The isoelectric point of the enzyme is pH 4.25. Studies of substrate specificity showed that the enzyme preferentially hydrolyzes dipeptides and dipeptidyl amides with L-proline or hydroxy-L-proline at the N-terminus. Longer chain substrates with N-terminal proline were not hydrolyzed.

A simple and rapid purification of kallikrein from rat submandibular gland

Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme.

Radioiodination of Proteins

Many different substances can be labeled by radioiodination. Such labeled molecules are of major importance in a variety of investigations, e.g., studies of intermediary metabolism, determinations of agonist and antagonist binding to receptors, quantitative measurements of physiologically active molecules in tissues and biological fluids, and so on. In most of those studies, it is necessary to measure very low concentrations of the particular substance and that in turn implies that it is essential to produce a radioactively labeled tracer molecule of high specific radioactivity. Such tracers, particularly in the case of polypeptides and proteins, can often be conveniently produced by radioiodination.

Purification of prokallikrein from bovine pancreas

A prokallikrein was isolated from bovine pancreas by a multi-step procedure involving gel filtration, hydrophobic interaction and anion-exchange chromatographies. The purification was initially monitored by measurement of the kinin-releasing activity of the activated zymogen. Later, when the pure prokallikrein had been isolated, a specific radioimmunoassay for the zymogen was set up and that was employed to provide estimates of 323-fold and 28% for the overall degree of purification and percentage recovery of prokallikrein. The relative molecular weight of prokallikrein was found to be 26,900 by SDS gel electrophoresis and its isoelectric point was established as pH 4.55.