2,5-Dihydroxyacetophenone: a matrix for highly sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of proteins using manual and automated preparation techniques.

2,5-Dihydroxyacetophenone (DHAP) is presented as a matrix which enables highly sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides, proteins and glycoproteins on AnchorChip targets. Depending on the protein, lower fmol amounts can be detected due to the increased homogeneity and concentration of the crystallization of the analyte/matrix mixture on the anchors. Best results could be generated in the mass range of 8-100 kDa. All sample/matrix preparation steps starting from mixing of DHAP matrix solution with sample solution to the transfer of the mixture to the MALDI-TOF target can be performed manually or automatically allowing low- and high-throughput analyses.

Glycogen phosphorylase BB in acute coronary syndromes.

The diagnosis of myocardial damage is preferably based on measurement of the cardiac-specific troponins. However, there is an emerging need for early, specific cardiac markers. One potential candidate is the glycogen phosphorylase BB isoenzyme (GPBB). We investigated the use of a new, commercially available GPBB ELISA assay in 61 patients presenting with an acute coronary syndrome (37 acute myocardial infarction, 24 unstable angina pectoris) in comparison to established cardiac markers such as troponin T, creatine kinase isoenzyme MB (CKMB) mass, and myoglobin. Blood samples were obtained on arrival, as well as 1, 2, 3, 4, 8, 12 and 24 h later. GPBB plasma concentrations were elevated in 90.9% of patients 1 h after onset of chest pain and increased to 100% at 4-5 h. Within the first 6 h, GPBB showed the highest sensitivity (95.5-100%) and high specificity (94-96%) compared to myoglobin (85-95% sensitivity) and CKMB mass (71.4-91.3% sensitivity). As expected, troponin T showed high specificity (100%) and sensitivity >95% later in the time course (>or=3 h). In un-stable angina pectoris patients, a very high rate of elevated GPBB was observed (93.9% at 3 h) compared to myoglobin (66.7%). Cardiac troponin T and CKMB were only elevated in 33.8% and 55.0% of these patients, respectively. In conclusion, GPBB is a promising marker for the early diagnosis of acute coronary syndromes and could probably act as a marker of ischemia. However, further studies on specificity and development of a fast, automated assay are necessary before GPBB can be recommended as a routine diagnostic tool.

[Changes in kinetic properties of pyridoxal-dependent enzymes during dietary vitamin B6 deficiency in rats]. / Izmenenie kineticheskikh svoistv piridoksal'zavisimykh fermentov pri alimentarnoi nedostatochnosti vitamina B6 u krys.

The erythrocyte aspartate aminotransferase and renal and intestinal glycogen phosphorylase activities in rats are determined as dependent on their provision with vitamin B6. It has been shown that the aspartate aminotransferase activity decreases and the shape of the aspartate concentration-activity curve changes in the vitamin B6-deficient animals. The B6 insufficiency does not affect the intestinal mucosa glycogen phosphorylase. However the renal phosphorylase activity decreases by 30 percent in the vitamin B6 deficient rats. It occurs due to changes in the affinity of phosphorylase A and B to glucose-1-phosphate but not to AMP. The activation of these investigated enzymes by exogenous pyridoxal phosphate reveals no essential differences between the vitamin B6-deficient and normal rats. The possible causes of the observed changes in the aspartate aminotransferase and phosphorylase activity are discussed.

[Glycogen phosphorylase from human leukocytes. Isolation and kinetic properties]. / Glikogenfosforilaza iz leikotsitov cheloveka. Vydelenie i kineticheskie svoistva.

Homogeneous glycogen phosphorylase from human leukocytes has been obtained. A one-step bioluminescent procedure for the enzyme activity assay has been developed. This method is based on a continuous recording of the product of the glycogen phosphorylase-catalyzed reaction using a coimmobilized multienzyme system (phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH:FMN oxidoreductase and bacterial luciferase). The method sensitivity is 10 times as high compared to earlier described methods. The Km values for glycogen (0.2 mg/ml) and phosphate (3.9 mM) at pH 7.9 were determined. AMP was shown to be the enzyme effector.

Immunoinhibition assay of the serum activity of human glycogen isophosphorylase BB in the diagnosis of the acute myocardial ischaemia.

Polyclonal rabbit anti-human isophosphorylases inhibit selectively the isoenzyme activities of the BB and MM isoenzyme of the human glycogen phosphorylase b with high sensitivity and without crossreactivities. On this basis a serological immunoinhibition assay of the BB isoenzyme in the range of 1-125 nmol s-1 l-1 is described. Preliminary clinical results indicate the BB isoenzyme as a sensitive and specific marker of the acute myocardial ischaemia including bypass surgery and suggest a substantial improvement of the differential diagnosis of infarction.

Liver glycogenosis caused by a defective phosphorylase system: hemolysate analysis.

Investigated were 24 cases of glycogenosis caused by a reduction in liver phosphorylase activity. The intravenous glucagon tolerance test could not discriminate between phosphorylase kinase deficiency [glycogen storage disease (GSD) IX] and phosphorylase deficiency (GSD VI). These two subgroups were distinguished by hemolysate enzyme assays: (1) GSD IX was characterized by a residual phosphorylase kinase activity, a low activation curve for endogenous phosphorylase b and increased amylo-1,6-glucosidase activity. (2) GSD VI was characterized by a normal or increased phosphorylase kinase activity, a slight activation of endogenous phosphorylase b and a normal amylo-1,6-glucosidase activity.