Edward S. Kline

Lactate dehydrogenase in rat mitochondria

Small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from rat heart, kidney, liver, and lymphocytes. Brain mitochondrial preparations were also isolated, but the results were inconclusive. A variety of cytosolic markers were used and it was found that essentially no cytosolic contamination was present except in brain preparations. A bacterial protease was used along with digitonin fractionation to determine localization of the mitochondrial LDH. Approximately 80% of the LDH activity associated with heart and kidney mitochondrial preparations was on the inside compared to about 40% for liver. Lymphocyte mitochondrial LDH activity was about 70% on the inside. Cytosolic LDH-5 preferentially adheres to outer mitochondrial membrane of liver, kidney, and heart. Agarose gel electrophoresis showed LDH isozymes in mitochondria qualitatively similar to that of the corresponding cytosol except in kidney mitochondrial preparations, where a specific electrophoretic band was found which did not correspond to any of the common LDH isozymes.

D- and L-Lactate Catabolism to CO2 in Rat Tissues

Abstract The current study was initiated in order to compare the rates of oxidative catabolism of D- and L-lactate in various rat tissues. Uniformly labeled D- or L-[14C]lactate was incubated at 37°C in a closed system with tissue homogenates in Krebs-Ringer phosphate buffer. Evolved 14CO2 was trapped in a center well containing a fluted filter paper saturated with strong base and the radioactivity determined. The ratio of L-lactate to D-lactate oxidation was greatest in brain, followed by kidney, heart, and liver. In liver the rate of oxidation of D-lactate exceeded that of L-lactate, in heart the rates were not significantly different and in the other two tissues L-lactate was oxidized more rapidly than D-lactate. These results indicate that the rate of D-lactate catabolism is considerable and is relatively greater than had been reported previously.

Evidence for polyphosphate in phosphorylated nonhistone nuclear proteins.

Structural studies of eucaryotic nuclear proteins have revealed the presence of bound polymeric phosphates. 32P-labeled and nonlabeled nonhistone nuclear proteins (NHPs) were isolated from rat liver nuclei and subjected to various controlled hydrolytic conditions. The analysis of protease-trypsin limit peptides revealed the presence of six phosphorylated, homogeneous fragments with phosphate/amino acid molar ratios greater than unity, ranging from 1.3 to 79. Alkaline beta elimination of phosphoester bonds released polymeric phosphates with chain lengths from 2 to over 200, as determined by using two-dimensional chromatographic analysis. The identity of these labeled polymeric phosphates was established to be polyphosphate by a number of criteria, including chromatographic mobility, gravimetric precipitation to constant specific activity, generation of orthophosphate on hydrolysis, and the determination of the delta H of hydrolysis of phosphoanhydride bonds. The evidence suggests that, in addition to the phosphomonoesters of serine and threonine, multiple phosphoanhydride linkages can result in the formation of polyphosphorylated NHPs. Previous investigators have demonstrated that exogenous, free polyphosphate causes destabilization of chromatin and enhancement of transcription in vitro. Although the function of the polyphosphorylated NHPs is currently unknown, such findings have possible functional implications with regard to the postulated role of NHPs as positive modifiers of gene expression.

Lactic Dehydrogenase Isozymes During Development of Azo Dye Tumors

Summary During the administration of the hepatocarcinogen 3‘-methyl-4-dimethyl-aminobenzene changes occurred in the distribution of lactic dehydrogenase isozymes in rat liver. This new pattern persisted in those animals which were maintained on the carcinogenic diet until tumors developed. If animals were removed from the carcinogenic diet early enough to prevent the ultimate formation of tumors the altered pattern soon reverted to “normal” in most cases. However, if animals were removed from the carcinogenic diet too late to prevent the ultimate formation of tumors the altered pattern was still in evidence after these animals developed tumors.

Species dependent effects of doxapram HCl on alcohol dehydrogenase activity

Abstract Doxapram HCl enhanced alcohol dehydrogenase activity of a rat liver extract. Little, if any, stimulation was observed when crystalline horse liver alcohol dehydrogenase was studied. In contrast, alcohol dehydrogenase activity in rabbit liver preparations was inhibited by the drug. The interesting species-dependent effect of this drug was further studied with respect to the electrophoretically separable forms of alcohol dehydrogenase. Comparative studies with lactic dehydrogenase indicated that this enzyme was not affected by doxapram HCl. Problems related to the identification of electrophoretically distinct forms of lactic dehydrogenase and alcohol dehydrogenase were discussed.

A Model System for Direct in vivo Measurement of Absorbed Nutrients in Portal Venous Blood

In order to investigate the in vivo effect of alcohol on intestinal absorption and on other factors which influence the rate of appearance of nutrients into the portal venous system, an experimental system has been developed in which separate cannulas have been implanted in the duodenum and portal vein of the rat. Following recovery from surgical procedures, unanesthetized animals may be administered nutrients directly into the duodenum, with and without alcohol, and the rate of appearance of nutrients in the portal system may be monitored by rapid sampling of the portal blood. Potential applicability of this experimental animal is demonstrated by the kinetics obtained with an amino acid, L-phenylalanine, both in the presence and the absence of alcohol.

Effects of proteolytic and thermal action on the activity of lactate dehydrogenase isozymes LD-1 and LD-5

Abstract L-Lactate dehydrogenase (LD) catalyzes the interconversion of pyruvate and lactate. Using a spectrophotometric assay to determine LD activity, incubation of rabbit, porcine, and bovine LD-1 and LD-5 isozymes with the protease subtilisin (Carlsberg) gave first-order degradation kinetics. Degradation half-lives were significantly lower for the LD-5 isozymes from the three species when incubated with subtilisin at temperatures from 4°C to 25°C. The energy involved in the degradation process, however, was not different. The activation energy for the conversion of pyruvate to lactate by LD-1 at pH 7.4 was significantly higher than that for LD-5 for all three species examined (P < 0.005). Thermocalorimetry showed that the LD-1 isozymes have both a higher mean temperature of denaturation and a higher heat uptake during the denaturation process than corresponding LD-5 forms. The results suggest that the LD-5 isozymes in the species studied are more metabolically efficient, whereas the LD-1 forms have greater structural stability.

Mitochondrial Lactate Dehydrogenase

The classical lactate dehydrogenase (LDH) catalyzes the interconversion of lactate and pyruvate:

Localization of l-lactate dehydrogenase in mitochondria

Pyruvate + NADH + {H^ + } \leftrightarrow L - lactate + NA{D^ + }\quad