Optimized conditions for determining activity concentration of alpha-amylase in serum, with 1,4-alpha-D-4-nitrophenylmaltoheptaoside as substrate.

We describe a method for measuring the catalytic activity of alpha-amylase (EC 3.2.1.1) in serum and urine, by use of a defined substrate: 1,4-alpha, D-4-nitrophenyl maltoheptaoside. We use a phosphate buffer of pH 7.10, containing chloride as activator and alpha-glucosidase (EC 3.2.1.20) as the auxiliary enzyme. After a lag phase of 4 min at 25 degrees C or 30 degrees C, or 3 min at 37 degrees C, the increase of absorption of 4-nitrophenol is measured at 410 nm or 405 nm. The pH value of the assay mixture is a compromise between optimum pH for the alpha-amylase reaction, shortest possible lag phase, and an acceptable absorptivity of 4-nitrophenol. Because the dissociation of 4-nitrophenol depends strongly on pH and temperature, we determined its absorptivity with various combinations of these variables in the assay. Heparin-treated plasma can be used, but not EDTA, fluoride, or citrate. Lipemia, hemoglobin less than or equal to mumol/L, bilirubin less than or equal to 170 mumol/L, glucose less than or equal to 100 mmol/L, and ascorbic acid less than or equal to 1 mmol/L of sample do not interfere in the assay.

Mechanism of action of human pancreatic and salivary alpha-amylase on alpha-4-nitrophenyl maltoheptaoside substrate.

We evaluated the enzymic mechanism by which alpha-4-nitrophenyl maltoheptaoside serves as a substrate for serum amylase (EC 3.2.1.1). Because polymeric substrates possess many potential sites of cleavage, the expression of enzymic activity as the number of micromoles of substrate transformed under defined conditions by an enzyme must be changed to "one microequivalent of group transformed." Therefore, we measured the activity of the isoenzymes of alpha-amylase with regard to suitable oligosaccharide substrates, which allowed us to express the catalytic activity in IUB units (U). By "high-performance" liquid chromatography we investigated the mechanism of human pancreatic and salivary alpha-amylase action, both alone and in combination with alpha-glucosidase (EC 3.2.1.20). On the basis of these results, we can describe exactly the entire reaction sequence and determine the stoichiometric coefficient of 4-nitrophenol within 0.02 mol/L (SD) produced under the assay conditions.

Action pattern of human pancreatic alpha-amylase on maltoheptaose, a substrate for determining alpha-amylase in serum.

An enzymatic assay for the determination of alpha-amylase in serum was developed which employed a soluble substrate, maltoheptaose, and a coupled enzymatic indicator reaction consisting of alpha-glucosidase and the hexokinase-glucose-6-phosphate dehydrogenase system. We used high-performance liquid chromatography (HPLC) to establish the action pattern of maltoheptaose under the test conditions: (A) the action pattern of alpha-amylase, (B) that of the combined action of alpha-amylase and alpha-glucosidase. Conductive to this effect was: the availability of pure maltoheptaose and human pancreatic alpha-amylase; the development of an adequate procedure for sample pretreatment (partition chromatography on a mixed-bed ion exchange) and of an HPLC system for separation of substrate and reaction products without interference from by products of the assay (partition chromatography on a cation-exchange column with acetonitrile-water); and the use of a new, very sensitive refractometric detector revealing sugar amounts as low as 40 ng. We derived the following stoichiometric equations: (see formula index).

[Two methods for the determination of working life tables and their problems]. / Verschiedene Verfahren zur Ermittlung von Erwerbspersonentafeln und ihre Problematik

PIP: 2 methods for the determination of tables of working life are presented in this paper. The 1st table is based on individual cohorts of working persons of the same age so that, altogether, a separate table of working life can be established for each of these cohorts. Detailed data are required for this method, and they are unavailable at this time. Thus, a less than perfect approach is presented which is based on Markov chains with homogenous transition probabilties for all working and nonworking individuals. No difference is being made between 1st employment and reemployment. The estimation with available data is possible. (author's)^ieng

[Synthesis of a fragment of the active center of the streptococcal proteinase (EC 3.4.22.10), VIII (author's transl)]. / Synthese einer Partialsequenz aus dem aktiven Zentrum der Streptokokken-Proteinase (EC 3.4.22.10), VIII

The synthesis of the protected tricosapeptide Z-Val-Lys(Z)-Pro-Gly-Glu(OBzl)-Gln-Ser-Phe-Val-Gly-Gln-Ala-Ala-Thr-Gly-His-Cys(MBzl)-Val-Ala-Thr-Ala-Thr-Ala-ONB is described. The tricosapeptide was built up from the decapeptide Z-Val-Lys(Z)-Pro-Gly-Glu(OBzl)-Gln-Ser-Phe-Val-Gly-ONp and the tridecapeptideester Gln-Ala-Ala-Thr-Gly-His-Cys(MBzl)-Val-Ala-Thr-Ala-Thr-Ala-ONB. A further nonadecapeptide with tert-butylmercapto protection of the SH group was also synthetized.