The medium-chain carnitine acyltransferase activity associated with rat liver microsomes is malonyl-CoA sensitive.

The data presented herein show that both rough and smooth endoplasmic reticulum contain a medium-chain/long-chain carnitine acyltransferase, designated as COT, that is strongly inhibited by malonyl-CoA. The average percentage inhibition by 17 microM malonyl-CoA for 25 preparations is 87.4 +/- 11.7, with nine preparations showing 100% inhibition; the concentrations of decanoyl-CoA and L-carnitine were 17 microM and 1.7 mM, respectively. The concentration of malonyl-CoA required for 50% inhibition is 5.3 microM. The microsomal medium-chain/long-chain carnitine acyltransferase is also strongly inhibited by etomoxiryl-CoA, with 0.6 microM etomoxiryl-CoA producing 50% inhibition. Although palmitoyl-CoA is a substrate at low concentrations, the enzyme is strongly inhibited by high concentrations of palmitoyl-CoA; 50% inhibition is produced by 11 microM palmitoyl-CoA. The microsomal medium-chain/long-chain carnitine acyltransferase is stable to freezing at -70 degrees C, but it is labile in Triton X-100 and octylglucoside. The inhibition by palmitoyl-CoA and the approximate 200-fold higher I50 for etomoxiryl-CoA clearly distinguish this enzyme from the outer form of mitochondrial carnitine palmitoyltransferase. The microsomal medium-chain/long-chain carnitine acyltransferase is not inhibited by antibody prepared against mitochondrial carnitine palmitoyltransferase, and it is only slightly inhibited by antibody prepared against peroxisomal carnitine octanoyltransferase. When purified peroxisomal enzyme is mixed with equal amounts of microsomal activity and the mixture is incubated with the antibody prepared against the peroxisomal enzyme, the amount of carnitine octanoyltransferase precipitated is equal to all of the peroxisomal carnitine octanoyltransferase plus a small amount of the microsomal activity. This demonstrates that the microsomal enzyme is antigenically different than either of the other liver carnitine acyltransferases that show medium-chain/long-chain transferase activity. These results indicate that medium-chain and long-chain acyl-CoA conversion to acylcarnitines by microsomes in the cytosolic compartment is also modulated by malonyl-CoA.

Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms.

A procedure was developed to determine the percentage of skeletal muscle actin and cardiac actin present in different striated muscle tissues. The method was applied to 2 mg of actin mixtures isolated from various origins. All samples show simultaneous expression of both striated muscle isoactins, with the cardiac actin being the major form (congruent to 80%) in 11-day-old chick embryonic leg muscle, decreasing to approximately 50% values in the late fetal stage of chicken, mouse, and in fused mouse muscle cell cultures and becoming the minor species (less than 5%) in adult skeletal muscle tissues. We also find a significant amount (up to 20%) of the skeletal muscle isoform in adult heart (ventricle) of porcine, bovine, and human origin and no differences in muscle actin ratios in human atrium and ventriculum cells. Similarly, no significant variation in the actin ratios was observed between a normal heart and a heart from a patient with hereditary obstructive myopathy. For those cells and tissues where comparison with levels of mRNA was possible we mostly find a good correlation between the relative ratios of expression of cardiac and skeletal actin proteins and mRNAs.

Sequential accumulation of mRNAs encoding different myosin heavy chain isoforms during skeletal muscle development in vivo detected with a recombinant plasmid identified as coding for an adult fast myosin heavy chain from mouse skeletal muscle.

In order to study developmental transitions of myosin heavy chain gene expression, we have cloned from newborn mouse skeletal muscle a recombinant plasmid (plasmid MHC 32) that contains an insertion coding for the COOH-terminal portion of an adult fast myosin heavy chain isoform of mouse skeletal muscle. By Northern blots and dot blots, it has been shown that the MHC 32 sequence reveals a broad cross-hybridization with RNA from different mammalian striated muscle tissues. Southern blots with mouse genomic DNA show only one homologous gene, but cross-hybridization at lower stringency to seven to eight different bands, some containing multiple genomic fragments, among which are probably the genes encoding the different striated muscle isoforms. S1 protection experiments with RNA from mouse skeletal muscle before and after birth demonstrate that plasmid MHC 32 is homologous to a major mRNA species of adult skeletal muscle. This adult mRNA is a predominant sequence within 5-6 days after birth. It begins to accumulate at 1-3 days; at the 18th day fetal stage, another major mRNA species is detected as partially homologous with the adult MHC 32 sequence. This fetal myosin heavy chain mRNA is still predominant at 1-3 days after birth, but is rapidly (by 5-6 days) replaced by the adult MHC sequence. There is thus a rapid transition after birth from fetal to adult skeletal muscle myosin heavy chain mRNA sequences.

Is the major Drosophila heat shock protein present in cells that have not been heat shocked?

When eukaryotic cells are exposed to elevated temperatures they respond by vigorously synthesizing a small group of proteins called the heat shock proteins. An essential element in defining the role of these proteins is determining whether they are unique to a stressed state or are also found in healthy, rapidly growing cells at normal temperatures. To date, there have been conflicting reports concerning the major heat-induced protein of Drosophila cells, HSP 70. We report the development of monoclonal antibodies specific for this protein. These antibodies were used to assay HSP 70 in cells incubated under different culture conditions. The protein was detectable in cells maintained at normal temperatures, but only when immunological techniques were pushed to the limits of their sensitivity. To test for the possibility that these cells contain a reservoir of protein in a cryptic antigenic state (i.e., waiting posttranslational modification for use at high temperature), we treated cells with cycloheximide or actinomycin D immediately before heat shock. HSP 70 was not detected in these cells. Finally, we tested for the presence of a reservoir of inactive messages by using a high stringency hybridization of 32P-labeled cloned gene sequences to electrophoretically separated RNAs. Although HSP 70 mRNA was detectable in rapidly growing cells, it was present at less than 1/1,000th the level achieved after induction.

The heat shock response is self-regulated at both the transcriptional and posttranscriptional levels.

When Drosophila cells are shifted from 25 degrees C to 37 degrees C, the synthesis of a small group of proteins (the heat shock proteins or HSPs) is rapidly induced, while most preexisting synthesis is repressed. On return to normal growing temperatures, synthesis of HSPs is gradually repressed and normal synthesis is restored. We show that production of HSP 70 (the major heat-induced protein in these cells) is quantitatively correlated with the degree of stress. The level of synthesis is controlled both transcriptionally and posttranscriptionally through repression of HSP 70 mRNA synthesis and destabilization of HSP 70 transcripts. These regulatory mechanisms depend upon the accumulation of the HSPs themselves; when the production of functional HSPs is blocked, HS transcription continues and HS mRNAs are stable, accumulating in vast quantities; if the block is released, a specific quantity of functional HSP must accumulate before HS transcription is repressed and preexisting HS mRNAs are destabilized. Evidence is also presented that indicates that the same quantity of HSP 70 is required to release the block in normal protein synthesis.

Heat shock and recovery are mediated by different translational mechanisms.

When Drosophila cells are shifted from 25 degrees C to 37 degrees C, protein synthesis is rapidly redirected from the complex pattern characteristic of normal growth to the simple pattern of heat shock proteins (HSPs). On return to 25 degrees C, synthesis of normal proteins is gradually reactivated and that of HSPs is repressed. In quantifying many different recovery experiments, we found that preexisting mRNAs always behaved as a cohort, with messages for different proteins returning to translation at the same rate. Heat shock mRNAs (HS mRNAs), on the other hand, never behaved as a cohort. Their repression was asynchronous, with translation of hsp70 always the first and translation of hsp82 always the last to be repressed. Although recovery times varied enormously (depending on the severity of the heat treatment), repression of hsp70 was always correlated with restoration of normal synthesis, suggesting a link between the two events, hsp70 repression was not simply due to competition with reactivated 25 degrees C mRNAs. A general decline in the translation efficiency of hsp70 mRNA was not observed. Instead, an increasing number of messages were translationally inactivated, while those remaining in the translational pool retained full ribosome loading. Unlike inactive 25 degrees C mRNAs, which are stable during heat shock, inactive HSP mRNAs are degraded during recovery.