James L. Conklin

Starch gel electrophoresis of lactic dehydrogenase from rat kidney.

Summary 1. An improved method for demonstration of lactic dehydrogenase after starch gel electrophoresis is described. This method employs a more sensitive tetrazolium and phenazine methosulfate. 2. Rat kidney contained 5 lactic dehydrogenase fractions which varied in cofactor requirements. DPN diaphorase was not demonstrable in any of these fractions, when reduced DPN was employed as the substrate, but was demonstrated in 2 additional bands. 3. The possibility is discussed that at least certain of the lactic dehydrogenase fractions from rat kidney exhibit electrophoretic heterogenity because of their association with various components of the respiratory system. We are deeply indebted to Mrs. Julia Ternak for technical assistance.

Phenazine Methosulfate; its Use in Evaluating Activity of Dehydrogenase Systems of Avian Liver

Lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and suecinate dehydrogenase were demonstrated in livers of 15-day chick embryos. The addition of phenazine methosulfate (PMS) to the LDH and MDH incubation mixtures reduced diformazan deposition in the liver epithelium but not in connective tissue. A 30 sec formalin fixation, absence of PMS, or the addition of sodium azide or potassium cyanide to the PMS-containing incubation mixtures facilitated formazan deposition. These results are explained by assuming that, in the absence of PMS, dehydrogenase activity is demonstrated via endogenous diaphorase. When PMS is present, Nitro BT reduction occurs within the incubation mixture. A side effect of the azide or cyanide is an interference with, the action of PMS, thus allowing diformazan deposition via the endogenous diaphorase when this is present in the tissue.

Subfractions of lactate dehydrogenase of the rat.

A survey of the lactate dehydrogenase (LDH) isozymes of 20 organs and tissues of the rat revealed a total of nine fractions exhibiting LDH activity after starch gel electrophoresis. Of the nine LDH fractions, eight were found in testis and lymph node, while the ninth fraction was unique to kidney. The occurrence of LDH fractions in excess of the usual five LDH isozymes is accounted for by two alternate proposals. One proposal is an extension of the present tetramer postulate of LDH structure, while the other is a consideration of the significance of heterozygosity in the albino rats employed in the study. Both hypotheses are plausible and should be considered in future studies of isozymes.

Development of Lactic Dehydrogenase Isozymes in the Chick Embryo.

Summary It was observed that 6 LDH isozymes were present in chick embryonic organs. Five of these isozymes occurred in developing liver while the 6th type was one of 3 isozymes found in brain and spleen. Brain and spleen exhibited only slight changes in isozyme content during maturation while more extensive changes occurred in liver. The demonstration of LDH isozymes of liver transplanted to the chorioallantoic membrane revealed that there was a reduction in the number of isozymes suggestive of a return to a less differentiated state.

ON THE HETEROGENEITY OF LACTIC DEHYDROGENASE

Yasummsni and! Ichikawa (J. Lab. & Clin. Med. 41: 296, 1953) recommmnusendcd the ninhydnin-Schiff reaction mis a histochemicmol test for proteins. Yet, in moo!el cxpenimssents smith various proteins only ali)umims svas colored (Burstone: J. Histocheni. 3: 32, 1955). When the nirshydnin-Schiff reaction sm-as applied to hunusan autopsy mmsatenial the intensity of staining wmus vanimuble and difficult to reproduce. The intensity of time reaction varied also with the hnnomuo! of ninshys!nin (Fisher, Emtstmams, Dajac). It wmos therefore deenned desirmobie to review penti nenst chenmuical literature to obtain i nfonmmsation whether or not the ninhydnin-Schiff ummethod can i)e coussic!ered suitai)le for the histochennical identificatious of proteinms. Accorc!insg to Virtmunen (Z. physiol. Chem. 266: 195, 1940), Van Slyke and coworkers (J. Biol. Cheat. 141: 627, 1941), and Greenstein and Winitz (Chemistry of the Amino Acids, Vol. 2, John Wiley & Sons, Inc., New York, 1961) the ninhydnin reaction is specific for free amino acids in that it requires the presenuce ins the free unconjugated state, of both the carboxyl and the neighboring ammmino group. Neither monsirso acids with a blocked cari)oxyl on mu i)iocked nunminso grommp react mmith ninhydnium. Pepticles and proteins also do not foruss noblehytles, I)ilt are isydrolyzed by nuinshydnium. Timcse chcmicmul dmutmo ins!icate thmot aldehydes s!eummonsstrmutec! by the mmininydnin-Schiff reactions more s!crivet! eitiser fromus free muusmiuuoacids still prcsenst ins paraffin-enusbedc!es! tissunes, or froums ausmi nso mucis!s split off the proteins ciumuinus during tneatusuennt mvith ni uuhvo!ri muTisoingh usmost ansuinno nocids are onuly sligint-ly soleble ins alcohol, time s-ornesponding nnext--lowcr noldehmydes derivet! fronsm nolmunsine, vnolinme, lemncine, isoleucimse mmmd l)imens lmulmunsi ne are very sob mimic Sunpportcs! l)Y USI HS Grant#R(’ 7303 ammo! i\Ieo!icmo! College of Gs-orgimo Professionunsl Ho-search Fmnnush (‘,ranmt #51 -79. (Hodgnmmanm et al. : Handbook of (‘hemistry and Physics. Chemical Hmubben Ponbi. Co., Cleveland, 1955). It sccnss therefore prol)noble that diffusion occurs during treatment of tissune sectionss -smiths ninhydnin in alcoholic solution for 16 hours or longer. Furthernusore, glyci ne , proli ne monsd hsydnoxyproline do not yield aidehydes (Van Slyke mind commorkers). Thus, these ausmino mocis!s, mm-iuich consstitute approximately 50% of the anuino acids in coilagen, can not be visualized mm-ith the ninhydnin-Schiff nsethod. Yet connective tissmne is often strongly colored. Landucci et at. (Recent -ldvances in Gelatin an(l Glue Resea,-ch, p. 62-67, Pengaumson Press, NewYork, 1958) reported thmut aldehydes are built into peptide chainss of collageus and other proteins. I)onning degradation of proteins these bonds betsveen momssino acids and nuldehvdes are i)roken and sommse of the resulting chsnoinus carry terumsinal alo!ehvde groimps . Under hmistos-isenmicmol condit ions coilagcns-svithout pretreat msmenmt-does not react with Schiff’s reagenut, but gelmuhine is mmtcnsseiv colored. However, it is nsot possible on the basis of these expenimmmcnts to cleterumminue whether or muot prefornued aldehydes l)lnu3 a role ins time nninmhydninu-Scimiff nemmction. It is commclmms!ec! that because of its ol)scinrc chenmicmi! bnosis, t hue irsherenut insmpossibihi ty to slensmonustrnote glycinme, proline minus! Isyo!roxypro!imme-t-hmot is, nspproxiussatcly 5(Y;: of the munsmimnonucioLs mmo-s)Ilmugemsmons! the probability of s!ifTusions norhifnocts, the uuinshvo!rinm-Ss,hiff nmmethoo! cnunn nuot i)c conmsicleresl a rehinuble iuistochemmmicnol mumet-huos! for the study of pnot-ei mutt.

MALATE DEHYDROGENASE ISOZYMES OF THE CHICK EMBRYO

Malate dehydrogenase fractions of the chick embryo were demonstrated after starch gel electrophoresis of homogenates of liver, brain and spleen. A total of seven malate dehydrogenase fractions were observed to occur in the chick embryo in an organ specific pattern. Treatment of the homogenates with urea, sodium chloride-sodium phosphate, and p-chloromercuribenzoate prior to electrophoresis revealed that only three distinct malate dehydrogenase-active proteins were presence. Two of these proteins exhibited properties similar to those previously reported for the supernatant malate dehydrogenase and mitochondrial malate dehydrogenase of other species. Becuase of the differing properties of chick malate and lactate dehydrogenase it is concluded that the molecular basis for malate dehydrogenase isozymes is different from that reported for lactate dehydrogenase isozymes.

Methods for the Demonstration of Lipid Applied to Compact Bone

Sections of compact bone were cut from the diaphysis of the femur, tibia, and humerus from dogs and monkeys. These sections were either ground thin and decalcified, or decalcified and subjected to frozen sectioning. Decalcification of the sections was effected by immersion in either Decal, 10% formic acid, 10% formic acid-sodium citrate (pH 4.5) or 20% aqueous EDTA. Sections were routinely stained with oil red O, Sudan black B, or Fettrot 7B. In addition, Nile blue A and phosphine 3R were also employed. Sections stained with phosphine were viewed with a fluorescence microscope. Control sections were extracted with lipid solvents prior to application of the staining procedures. The results indicate that lipid is present in compact bone within the osteocytes, lacunae, canaliculi, and organic matrix. The significance of the lipid in these sites, particularly extracellularly, is unknown.