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A filter paper assay was developed for measuring the activity of transamidating enzymes by the incorporation of radioactive amine substrates into proteins. The method is analogous to those generally employed for monitoring the enzymic coupling of isotopic groups to proteins, such as in the ATP-dependent phosphorylation of casein or histone. Use of minimal quantities of reagents, as well as simplicity of handling a large number of test samples, are distinct advantages of this technique. The guinea pig liver transglutaminase-catalyzed incorporation of C14-histamine and C14-putrescine into β-lactoglobulin and casein were studied. The apparent Michaelis kinetics of the reactions permitted evaluation of Km,app's for both isotopic substrates and competitive inhibition constants for nonradioactive amines were also obtained. In contrast to transglutaminase, the fibrinoligase—generated in human plasma from Factor XIII by the addition of thrombin—could not utilize β-lactoglobulin to an appreciable extent. Hence experiments had to be restricted to casein as acceptor protein. Apparent Michaelis constants for the isotopic substrates and inhibition constants for nonradioactive amines could be readily measured also in the complex plasma system. Finally, details of a rapid filter paper assay are described for measuring Factor XIII levels in human plasma by measuring the incorporation of C14-putrescine, at close to saturating concentrations, into casein.

A rapid filter paper disk assay for cyclic AMP based on the activation of a cyclic AMP dependent protein kinase from rabbit skeletal muscle is described. This procedure permits determination of as little as 0.2 picomoles of cyclic AMP. In addition this procedure employs ATP-γ-33P instead of ATP- γ-32P.

We describe a method for the assay of adenyl cyclase in whole tissue homogenates. Adenosine 3′:5′-cyclic monophosphate (cAMP) formed from α-32P-, 14C- or 3H-labeled adenosine 5′-triphosphate (ATP) substrate is isolated from all known ATP metabolites and an unknown metabolite by electrophoresis in 1% sodium borate for 40 min, followed by overnight descending chromatography in 95% ethanol:1 m ammonium acetate (70:30). The purity of the cAMP isolated is established by chromatographic techniques as well as by utilizing a purified cyclic nucleotide phosphodiesterase. The method described here also makes possible the measurement of phosphodiesterase activity in homogenates. It is rapid enough to allow routine assay of 180 samples per day, although the number of samples processed depends on the number of electrophoretic and chromatographic units available.

Drug derivatives prepared by injection of a mixture of drug and reagent into a gas chromatograph can be collected and subjected to elevated temperature reversed-phase paper chromatography. This procedure provides additional parameters which have been found useful in identifying drugs in samples of blood, urine and liver.

A rapid, simple, sensitive and reproducible filter-paper assay was developed to follow the glucose transfer from uridine diphosphate [ 14 C]glucose to protein or protein-bound oligosaccharides. This method could be useful for following the formation of any glycoprotein. Rectangles of trichloroacetic acid-treated filter paper are introduced into a test tube containing the reaction mixture. The papers are submitted to a batch washing-procedure that ensures the removal of the compounds that are not trichloroacetic acid-precipitable. After scintillation counting, the paper can be reused to study the acid lability of the glucoprotein.

Conventional usage of the filter-paper disc technique following in vivo hepatic incorporation of certain labeled pyrimidine and purine RNA precursors resulted in the presence of radioactivity on the filter disc in species other than RNA. Thus the filter paper disc technique, conventionally conducted, may be misleading. To eliminate artifacts, it is proposed that replicate filter discs be processed, one group passing through the usual cold TCA washings only, while a parallel group of replicate discs are heated for 20 min at 90°C in 5% TCA to remove RNA. True RNA radioactivity is the difference of counts from the cold TCA precipitable material and the radioactivity remaining after hot TCA extraction.

A simple procedure is described for the storage of drugs extracted from biological material. The drugs are spotted on filter paper and can be extracted after several years without serious losses

IT has been shown by paper electrophoresis that xanthine dehydrogenase activity is bound to the globulin fractions of rat serum1. Using differential centrifugation, it was demonstrated that xanthine oxidase and xanthine dehydrogenase activities are present in the supernatant fluid corresponding to the ‘cell sap’ containing the soluble proteins of the rat liver2.

Transverse sections of linear partition chromatograms on filter paper when eluted with basal medium for Lactobacillus casei showed Wilson Liver Fraction L to contain at least two growth stimulating factors for this organism. Three growth stimulating factors were demonstrable by this technique in trypsin digest of casein in contrast to dried yeast solubles which contained but one such factor. The growth stimulating factor from yeast was separated and partially purified. Treatment with strong acid suggested the peptide nature of this material. Chromatographic evidence of the presence of glycine, leucine, aspartic acid, and glutamic acid was obtained from the acid hydrolyzate of the yeast factor.

A modified method for enzymic synthesis of UDP-14C-glucose in high yield is described. Labeled UDPglucose is isolated by high-voltage paper electrophoresis after an alkaline phosphatase treatment of the reaction products. The synthesis gives high yields and is reproducible, and the isolation of radioactive UDPglucose from the electrophoresis is quantitative. A new assay for UDPglucose: glycogen α-4-glucosyltransferase (EC 2.4.1.11) is also described. Enzyme reaction mixtures are spotted directly on filter paper squares and washed with 66% ( v v ) ethanol, and 14C-glycogen is counted in a liquid scintillation spectrometer. The method is accurate for all transferase preparations tested if corrections are applied for sample quenching. Assay time is greatly reduced and large numbers of assays can be processed with very little increase in assay time. The assay is also readily applicable to the determination of transferase-I kinase activity. It was also found that stopping the transferase-catalyzed reaction by precipitation of trichloroacetic acid insoluble protein resulted in a loss of radioactive glycogen. When crude preparations of transferase were assayed, up to 20% of the radioactivity bound to glycogen was precipitated by 6% trichloroacetic acid.

In a program of clinical trials of enzyme-activated nitrogen mustard drugs in human cancer patients, it was desirable that the “activating enzymes” should be assayed in small biopsies of the tumors to aid in the choice of a suitable drug. Biochemical “test tube” methods of enzyme assay were sometimes time consuming and laborious and often required fairly large quantities of tissue, as when several enzymes had to be assayed in a tissue sample. In the present work, these difficulties were overcome by incubating substrates for esterase, amidase, and aminopeptidase with a single sample of tumor homogenate (containing 10 mg wet weight of tissue). The hydrolysis products were separated and estimated by a rapid method of paper chromatography and the total time involved in obtaining an assay of the three enzyme activities was not more than 3 hr.

A rapid, sensitive and simple procedure for the assay of l -methionine- S -adenosyltransferase based on the use of phosphocellulose ion-exchange paper is presented. The analytical procedure may be generally useful for all enzymes where there is a large cationic charge difference between the substrates and products. An application of a simpllified counting procedure for radioisotopes on solid supports is presented.

Mediante elettroforesi su carta si dimostra la presenza nei lisati di mitocondri di fegato di ratto di almeno 5 componenti proteici. Le colorazioni specifiche dimostrano la presenza di 3 frazioni lipoproteiche e di altrettante glicoproteiche. I lisati dei mitocondri vennero ottenuti qer trattamento delle particelle con Triton X-100.

Gli autori hanno studiato il comportamento delle proteine e delle glicoproteine solubili del fegato rigenerante di ratto mediante l'elettroforesi su carta. La colorazione con Amidoschwarz mostra che il comportamento elettroforetico delle proteine di fegato rigenerante e praticamente sovrapponibile a quello del fegato normale. La colorazione secondoKoiw eGronwall dimostra che le glicoproteine diminuiscono nei primi giorni dopo l'atto operatorio, tornando pero alla normalita circa 30 giorni dopo l'epatectomia.

We studied the alkaline α-naphthyl phosphatase isoenzymes from livers, kidneys, small intestines and testes of the rat with DEAE-paper chromatography (developing medium: o.i M sodium barbital containing 0.3 M NaCl). A histochemical assay procedure visualized several bands whose patterns were much different in the various organs. Among enzyme preparations, testicular enzyme changed its Chromatographie pattern after a month's standing at 0–2°.

Viene studiato il comportamento elettroforetico su carta delle proteine cellulari di fegato normale ed in degenerazione vacuolare e grassa. Nei ferogrammi di fegati in degenerazione vacuolare si ha la comparsa di una banda di tipo albuminico, che non compare nei ferogrammi normali. I ferogrammi di fegati in degenerazione grassa si rivelano piu semplici di quelli normali per la mancanza di una banda di tipo globulinico. Questi risultati vengono brevemente discussi.

THE electrophoretic analysis by the ordinary Tiselius apparatus of the soluble proteins isolated from rat liver has already been described1–3; but the constituent components reported by these authors differed one from the other.

A sialoglycopeptide is isolated from human pregnancy urine after gel filtration, ion-exchange chromatography, paper chromatography, and high voltage paper electrophoresis. It contains serine, N-acetyl-galactosamine, galactose, sialic acid (1-1-1-2). Structural studies show that the carbohydrate moiety is likely O-glycosidically linked to serine, and related to MN blood group structures. However this glycopeptide does not exhibit any cross antigenic reactivity by the hemagglutination assay using anti-N, anti-M rabbit immune sera or anti-N lectin from Vicia Graminea.