Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.     

Streptozotocin-induced diabetes produces alterations in adenylate cyclase in rat cerebrum, cerebral microvessels and retina

Eight weeks following streptozotocin-induced diabetes mellitus in rats, the sensitivity of adenylate cyclase to dopamine (DA) and norepinephrine (NE) was reduced in homogenates of retina. Furthermore, the activation of adenylate cyclase in cerebral microvessels (capillaries) by NE, 5'-guanylyl imidodiphosphate (alone or with NE) and forskolin was reduced in diabetic rats versus appropriate controls. In diabetic rats enzyme sensitivity to only NE was attenuated in homogenates of cerebral cortex and cortical piaarachnoid. No differences between controls and diabetics were noted with respect to guanylate cyclase or cyclic AMP phosphodiesterases. The damage observed in retina and microvessels may play an important pathogenic role in diabetes-induced blindness and stroke.     

The diabetic Zucker fatty rat

A noninsulin-dependent diabetes appeared in fatty rats in our Zucker rat colony. A breeding program yielded a genetic pattern of diabetes consistent with a dominant gene not closely linked to the fatty gene. Fatty males were more frequently affected than fatty females. Since no markers could be identified for heterozygous carriers and since affected fatty rats were 6 months old when diabetes appeared, the diabetic trait could not be sustained in our small colony. Glucose tolerance tests showed that the diabetic fatty rats had little increase in plasma insulin concentration after a glucose load was administered. Plasma insulin concentrations were unchanged relative to control fatty rats. Percentage body fat and plasma triglyceride values were decreased in fatty diabetic rats relative to control fatty rats, however, consistent with insulin resistance in fat and liver. The content of pancreatic insulin was markedly decreased in the diabetic fatty rat relative to either the ad libitum or diet-restricted fatty rats. The occurrence of a genetically based diabetes in a normally outbred colony underscores the importance of genetic traits that interact with obesity in determining diabetes in rodent models.     

On the structure of the iron-sulfur complex in the two-iron ferredoxins

Recent spectroscopic and magnetic susceptibility studies of the iron center in the two-iron ferredoxins provide criteria which any model for the iron-sulfur complex in these proteins must satisfy. These criteria are most stringent for parsley and spinach ferredoxin: the reduced proteins contain a high-spin ferric atom antiferromagnetically exchange-coupled (presumably via sulfide bridging ligands) to a high-spin ferrous atom. In the oxidized proteins the iron atoms are antiferromagnetically spin-coupled, high-spin ferric atoms. Arguments are given to substantiate the claim that the ferrous atom in the reduced protein is ligated by four sulfur atoms in a distorted tetrahedral configuration: two are the bridging sulfides, two are cysteinyl sulfurs. A treatment of proton contact shifts based upon the above model is pertinent to proton magnetic resonance data already available and provides a means to identify directly the ligands at both iron atoms via further PMR experiments.     

DIVISION IN THE DINOFLAGELLATE GYRODINIUM COHNII (SCHILLER) : A New Type of Nuclear Reproduction

Dinoflagellates are of interest because their chromosomes resemble the nucleoplasm of prokaryotes both chemically and ultrastructurally. We have studied nuclear division in the dinoflagellate Gyrodinium cohnii (Schiller), using cells obtained from cultures undergoing phasic growth. Electron micrographs of serial sections were used to prepare three-dimensional reconstructions of nuclei and chromosomes at various stages of nuclear division. During division, a complex process of invagination of the intact nuclear envelope takes place at one side of the nucleus and results in the formation of parallel cylindrical cytoplasmic channels through the nucleus. These invaginations contain bundles of microtubules, and each of the bundles comes to lie in the cytoplasm of a cylindrical channel. Nuclear constriction occurs perpendicular to these channels without displacement of the microtubules. There are no associations between chromosomes and the cytoplasmic microtubules. In dividing cells most chromosomes become V-shaped, and the apices of the V's make contact with the membrane surrounding cytoplasmic channels. It is proposed that the membrane surrounding cytoplasmic channels in the dividing nucleus may be involved in the separation of daughter chromosomes. Thus, dinoflagellates may resemble prokaryotes in the manner of genophore separation as well as in genophore chemistry and ultrastructure.