The effect of heat shock on 20S/26S proteasomes.

We have studied the consequences of heat shock on 20S/26S proteasome activity and activation, the proteasomal subunit composition, proteasome assembly, subunit mRNA stability as well as on the intracellular distribution of proteasomes. Our data show that heat shock locks 20S proteasomes in their latent inactive state and impairs further activation of the 26S proteasome by ATP. Proteasome mRNA levels are decreased after heat shock and the assembly of the proteasome complex is inhibited. Heat shock also induces a rapid reorganisation of the cellular distribution of the proteasome which appears to be connected with proteasome activity and the change of the cellular architecture after heat shock.

Identification of a novel ATP-dependent proteolytic activity in mitochondrial intermembrane space.

The formation of primary amines via proteolysis was monitored in isolated rat liver, kidney cortex and heart mitochondria in the presence and in the absence of ATP. The highest proteolytic activity was detected in kidney cortex mitochondria with about 120 nmoles primary amines/hour x mg protein. The formation rates of liver mitochondria amounted to about 100 nmoles primary amines/hour x mg protein and in heart mitochondria about 60 nmoles primary amines/hour x mg protein. In all mitochondria investigated an ATP-dependent proteolysis of 20-40 nmoles primary amines/hour x mg protein was detected. The effects of various protease inhibitors were tested in rat liver mitochondria and thiol-specific reagents showed a 35-70% inhibition. The ATP stimulable portion of proteolysis was blocked by hemin, a known inhibitor of ATP-dependent proteases. The localization of the proteolytic activity was tested by fractionation of the compartments of rat liver mitochondria using the flourogenic peptide suc-Leu-Leu-Val-Tyr-MCA as substrate. About 90% of the ATP-dependent peptide cleavage activity were found in the mitochondrial intermembrane space. The characteristics of the enzyme were compared to those of other known mitochondrial ATP-dependent proteases and it was concluded that it represents a novel proteolytic system of the intermembrane space.

Influence of erythropoietin treatment on 2,3-bisphosphoglycerate and O2-affinity of red blood cells in children with renal anemia.

In children with renal anemia the concentration of DPG of red blood cells and the O2-affinity of hemoglobin (P50) were determined before and under treatment with EPO. Before treatment the concentration of DPG was 6.25 mmoles/l cells and the P50 28.9 Torr. A rise of the hematocrit to about 0.30 was accompanied by an increase of DPG to 7.95 mmoles/l cells and of P50 to 32.1 Torr, respectively. Thus improvement of renal anemia by stimulation of erythropoiesis by means of EPO was accompanied by a decreased O2-affinity of hemoglobin and therefore by an additionally improved O2-supply to the tissues.

Catabolism of adenine nucleotides in rabbit blood cells.

The present study describes the effects of the inhibition of adenosine deaminase and of adenosine kinase on reticulocytes and erythrocytes of rabbits. Both in erythrocytes and in reticulocytes the degradation of adenine nucleotides proceeds via AMP----IMP----inosine----hypoxanthine. Under approximately physiological conditions the rate of degradation amounts in erythrocytes to 23 mumoles/l cells.h and in reticulocytes to 331 mumoles/l cells.h, respectively. In erythrocytes the formation of hypoxanthine corresponds closely to the degradation of adenine nucleotides in reticulocytes; the formation of hypoxanthine seems to exceed the degradation presumably mainly due to RNA degradation. Parallel to the primary deamination of AMP there is a primary dephosphorylation to adenosine of about 60 mumoles/l cells.h in erythrocytes and about 300 mumoles/l cells.h in reticulocytes. This pathway does not provide, however, any measurable contribution to the formation of hypoxanthine, because the adenosine formed is rephosphorylated via adenosine kinase almost completely.

ATP-dependent peptide release from mitochondria of reticulocytes.

ATP-dependent release of TCA-precipitable peptides from mitochondria-containing stroma (MCS) is described. The process is independent of ubiquitin, but is sensitive to hemin and to heat treatment. Neither chloramphenicol nor EGTA inhibit. 50% of the activity is dependent on charged tRNA. The peptides released from MCS possess a molecular mass of about 1-5 kDa and are degraded to TCA-soluble compounds by a cytosolic protease system (fraction II) without ubiquitin.

Effect of substrate heat-denaturation on ATP- and ubiquitin-dependent proteolysis.

The susceptibility of human serum albumin (HSA) to degradation by the ATP- and ubiquitin-dependent proteolytic system is enhanced by heat-denaturation. The steady-state levels of formed ubiquitin conjugates, however, are equal with denatured and native HSA. In contrast, both the hemin sensitive Pi release and proteolysis with native HSA are less than one fourth of that with denatured HSA. The rate limiting step for the degradation of HSA by the ATP- and ubiquitin-dependent proteolytic system must be beyond the ubiquitin conjugation steps. Differences concerning the effects of heat denaturation of mitochondria-containing stroma and HSA on ATP- and ubiquitin-dependent proteolysis are discussed.

Kinetic studies on the ATP- and ubiquitin-dependent proteolytic system of reticulocytes.

Half-maximal rate of the ATP- and ubiquitin-dependent proteolysis of rabbit reticulocytes, determined in a cell-free system, require 0.5 mg/ml of mitochondria-containing stroma (MCS) protein at a fixed ATP concentration or 100 microM ATP at a fixed protein concentration. 20 microM hemin caused 50% inhibition of degradation of MCS.